Mouse Bone Marrow Cells Research Articles

Proteomic Screening Identifies Megakaryocyte Derived PF4/Cxcl4 As a Critical Driver of Myelofibrosis

Background: Despite advances in understanding the genomic landscape of Myeloproliferative Neoplasms (MPNs), the precise pathological mechanisms driving fibrotic progression in MPNs remain incompletely understood. Previous research has shown that Megakaryocytes (Mks) play a role in MPN pathology by promoting inflammation and extracellular matrix (ECM) deposition by activated stromal cells. However, the specific factors derived from Mks that contribute to the inflammatory milieu and myelofibrosis progression are not well defined. Aims: To investigate the events that drive bone marrow (BM) fibrosis due to abnormal megakaryocyte-stromal crosstalk, we employed mass spectrometry-based proteomics in a myelofibrosis-like mouse model. Our goal was to identify differentially expressed proteins involving Mks, platelets and BM cells in fibrotic versus non-fibrotic conditions. Methods: We induced myelofibrosis in mice through repeated injections of supra-pharmacological doses of the thrombopoietin-mimetic Romiplostim (TPO high). Subsequently, we isolated Mks, BM cells and platelets from TPO high and control mice (injected with saline) and analyzed protein extracts using LC-MS/MS assay. Cytokine levels in plasma and BM cell-free fluids of treated mice were assessed using cytokine arrays. To evaluate fibrosis-related markers, we analyzed the expression of myofibroblasts markers (a-SMA, Vimentin) and ECM synthesis (Fibronectin, Type III Collagen) in mouse embryonic fibroblasts (mEFs) and human BM mesenchymal stromal cells after stimulation with recombinant PF4 using western blot. mEFs were co-cultured with BM-derived Mks in direct and indirect co-culture systems in the presence of increasing TPO concentrations. In some experiments, Mks were transfected with siRNA specifically targeting CXCL4 gene or control siRNA before co-culture with mEFs. Additionally, we quantified plasma PF4/Cxcl4 levels using ELISA assay in a large cohort of MPN patients and normal controls. We also sorted CD41/CD61 Mks from BM aspirates of MPN patients and evaluated CXCL4 mRNA levels through qPCR. Results: We observed that signaling pathways related to cytoskeletal reorganization, cell adhesion and inflammation, were commonly activated in Mks, platelets and BM cells of TPO high mice compared to control mice. The MSP-RON signaling pathway and the Unfolded Protein Response were specifically activated in Mks and platelets, respectively. Among the differentially expressed proteins,the chemokine PF4/Cxcl4 was up regulated exclusively in the proteasomes of the TPO high mice. We confirmed Increased levels of PF4/Cxcl4 in plasma and BM cell-free fluids of fibrotic mice using cytokine arrays. In vitro stimulation of BM-derived Mks with high TPO concentrations resulted in increased synthesis and secretion of PF4/Cxcl4. Recombinant PF4/Cxcl4 was rapidly internalized through surface glycosaminoglycans by mEFs and human BM mesenchymal stromal cells, leading to myofibroblast differentiation. Importantly, we demonstrated that these mechanisms were interconnected during Mk-mEF cross-talk in co-culture systems with high TPO concentrations. Genetic down-regulation of CXCL4 in Mks, prior to co-culture in TPO-saturated conditions, mitigated the profibrotic phenotype of mEFs in terms of ECM synthesis and myofibroblast markers expression. Finally, we found higher plasma levels of circulating PF4/Cxcl4 and increased expression in BM-sorted Mks in patients with MPNs. Conclusions: Our findings identify a crucial role of Mk derived PF4 in the fibrosis progression of MPNs, providing further support for the potential therapeutic strategy of neutralizing PF4.

Tasquinimod Improves Erythropoiesis and Mitigates Bone Loss in Myelodysplastic Mice

Myelodysplastic neoplasms (MDS) are characterized by ineffective hematopoiesis, peripheral cytopenia, and a risk of transforming into acute myeloid leukemia (AML). Interestingly, MDS is also associated with an increased prevalence of osteoporosis. Studies have suggested that dysregulated innate immune responses and a proinflammatory bone marrow (BM) microenvironment play significant roles in the pathogenesis of MDS. Targeting inflammatory pathways, especially in low-risk MDS patients, presents a potential strategy to modulate the progression of the disease. One such candidate is Tasquinimod (TASQ, ABR-215050, Active Biotech AB), a small-molecule oral inhibitor and second-generation quinoline-3-carboxamide compound. TASQ binds to the inflammatory protein S100A9 and inhibits its interaction with the proinflammatory toll-like receptor 4, thereby displaying both immunomodulatory and antitumor properties. Our in vitro data provided evidence for a rescuing effect of TASQ on inflammatory signal-primed MDS stromal cells, leading to enhanced hematopoietic support. This study aims to validate these findings using a transgenic MDS mouse model expressing the NUP98/HOXD13 (NHD13) fusion protein in hematopoietic cells. NHD13 mice, generated on a C57BL/6J background, were obtained from Jackson Laboratory. TASQ was administered orally by adding it to the drinking water at a dose of 30 mg/kg. Both transgenic and littermate control (WT) mice (12 weeks old, n=5-10 per group) were treated with or without TASQ for 12 weeks. Throughout the experiment, peripheral blood cell counts were measured every 4 weeks using Sysmex analysis. In the final analysis, primary BM cells were isolated from the femora, and the number of lineage-negative, sca-1-positive, and c-kit-positive (LSK) cells, as well as erythroid differentiation, were assessed using flow cytometry. Additionally, bone volume, trabecular number, and thickness were analyzed in femora using µCT. Transgenic NHD13 mice displayed MDS-like characteristics, including peripheral cytopenias, dysplasia in blood and BM cells, and microstructural abnormalities in the bone microenvironment. As expected, hemoglobin levels (Hb) and red blood cell (RBC) counts were lower in NHD13 mice compared to WT mice [Hb: 8.9 vs. 9.5 mmol/L; RBC: 9.5 vs. 10.3x1012/L] and showed a consistent decrease in the untreated group from 12 to 24 weeks. Notably, treatment with TASQ significantly improved red blood cell counts in NHD13 mice [+14%; p<0.01] after 24 weeks, while having no effect on WT mice. White blood cells were significantly decreased in WT mice after 4 weeks of TASQ treatment [-19%; p<0.001] until the end of the experiment [-36%; p<0.001]. In contrast, in transgenic mice, the lower white blood cell counts [-76%; p<0.001] were not further diminished. Interestingly, TASQ led to an elevation in neutrophil counts. The platelet count was not affected by the treatment in both transgenic and WT mice. Using flow cytometry, an increase in the total number of LSK cells was detected in the BM of WT mice after TASQ treatment. However, no increase was observed in NHD13 mice, which already had lower LSK numbers compared to WT controls. Late-stage erythropoiesis was slightly increased in both WT and transgenic mice after TASQ treatment, whereas early erythroid differentiation was unaffected or even decreased by TASQ. Moreover, BM cells were used for colony-forming assays ex vivo. NHD13 mice had a lower erythroid differentiation potential compared to WT mice, which was improved after TASQ treatment. To assess the potential influence of the microenvironment, adherent BM cells were assayed for their CFU-F forming capacity. BM cells of transgenic mice exhibited a slightly lower CFU-F forming capacity compared to WT mice, which was partially reversed by TASQ treatment. Finally, the bone microarchitecture was analyzed. NHD13 mice showed a lower bone volume [-43%; p<0.01] compared to WT mice. Notably, treatment with TASQ abolished the bone loss in NHD13 mice by increasing the trabecular number [+35% p<0.001], whereas the trabecular thickness was not affected. In conclusion, this study provides the first evidence for an in vivo effect of TASQ in a murine model of MDS, suggesting improved erythropoiesis and positive effects on the bone phenotype. These results warrant further investigations of TASQ in human trials, particularly in MDS patients in whom anemia and bone loss often coexist.

Novel Hypomethylating Agent Ntx-301 Exhibits Anti-Leukemia Activity in Venetoclax-Resistant and TP53-Mutant AML

The FDA approved Bcl-2 inhibitor venetoclax/hypomethylation agent (HMA) combination has greatly improved outcomes in patients with AML. However, despite of high response rates, the median overall survival is only 14.7 months and 2.5 months post relapse. Novel therapies in therapy-resistant AML are urgently needed. Multiple mechanisms contribute to resistance to venetoclax therapies including mutations of oncogenic kinases, leading to constitutive activation of the FLT3, SHP2, and RAS signaling cascade and upregulation of antiapoptotic Bcl-2 proteins, and mutations of TP53. We here investigate the therapeutic efficacy of NTX-301 (5-aza-thio 2'-deoxycytidine) (PinotBio, Inc.), a novel HMA in venetoclax-resistant and TP53-mutant AML. NTX-301 greatly reduced the levels of DNMT1 and decreased viability in various AML cells, markedly more effective than 5-azacytide (5-Aza). NTX-301 decreased Mcl-1 and effectively induced cell death in AML cells overexpressing Mcl-1 and Bcl-2A1, both of which are upregulated by various kinases and resistant factors to venetoclax; in AML cells with intrinsic or acquired resistance to venetoclax; and in AML cells and stem/progenitor cells obtained from patients who were resistant/relapsed from venetoclax-based therapies. Furthermore, cell death induction in aforementioned cells was synergistically enhanced when NTX-301 was combined with venetoclax. NTX-301 alone or venetoclax/NTX-301 combination had minimal activity in normal bone marrow cells and bone marrow CD34 + stem/progenitor cells. Importantly, using a PDX model developed from an AML patient who relapsed from venetoclax/HMA, NTX-301 alone demonstrated great anti-leukemia activity and significantly extended survival (167 vs 114 days of control, P=0.0001), while venetoclax or venetoclax/5-Aza did not. CyTOF analysis of mouse bone marrow cells collected at the end of 5 week treatment showed that NTX-301 and even more so NTX-301/venetoclax effectively decreased CD45 + leukemia blast cells and CD34 +CD38 +/CD34 +CD38 - AML stem/progenitor cells, while venetoclax or venetoclax/5-Aza had not effects. We observed that NTX-301 activated p53 in TP53 wild-type AML cells. We also observed that NTX-301 increased p73 and decreased MDM2 in TP53-mutant cells and showed activities in TP53-mutant leukemia cells. NTX-301 and venetoclax combination synergistically induced cell death and decreased viable cells in AML cells and stem/progenitor cells with TP53 mutations. Additionally, NTX-301 treatment increased caspase-8 and cleaved-caspase-8 in AML cells independent of TP53 mutation status, consistence with reports showing caspase-8 hypermethylation in AML and supporting that activation of caspase-8-mediated extrinsic apoptosis as a mechanism of NTX-301 action. In conclusion, our data suggest that NTX-301 has more potent anti-leukemia activities compared to current HMA in clinic and synergizes with venetoclax in venetoclax-resistance and TP53-mutant AML and AML stem/progenitor cells, and warrants clinical evaluation

The DPP4 Dependent Transcriptome Identifies Pathways to Improve Mouse Hematopoietic Cell Function

Hematopoietic cell transplantation (HCT) is a curative treatment for hematologic disorders wherein healthy donor hematopoietic stem (HSCs) and progenitor cells (HPCs) collected from bone marrow (BM), mobilized peripheral blood (PB) or umbilical cord blood (CB) reconstitute an ablated hematopoietic system. There is a strong need to enhance functional competency of HSCs/HPCs for HCT to prevent complications like graft versus host disease (GVHD), relapse, and graft failure. Dipeptidyl peptidase 4 (DPP4) is a protein that cleaves N-terminal dipeptides from hematopoietic factors, impacting their function. Inhibition of DPP4 improves HSC/HPC competency in mouse models of HCT and clinically speeds neutrophil recovery after CB transplantation and reduces incidences of GVHD after PB transplantation. Thus, DPP4 inhibition is a viable component of a multi-pronged strategy to improve engraftment and prevent GVHD. While DPP4 inhibition improved early neutrophil recovery in clinical trials, the effects were modest, required high dose treatment, and effects on platelet recovery were unclear. Examining mechanisms by which DPP4 inhibition drives HSC/HPC functional competency may yield insight into targets that can enhance DPP4 driven improvements to HCT. We harvested BM from mice treated with the DPP4 inhibitor Diprotin A (DPA) and sorted immunophenotypically defined HSCs, multipotent progenitors (MPPs), common myeloid progenitors (CMPs), and granulocyte-macrophage progenitors (GMPs) for transcriptomic analysis. Linear modeling to examine differential gene expression controlling for cell type revealed that DPP4 inhibition gave strong enrichment of gene programs associated with mitochondrial function. Using a mitochondrial stress test and the Seahorse metabolic flux analyzer, we found that lineage depleted mouse BM cells treated ex vivo with DPA had lower basal oxygen consumption rates and spare respiratory capacity. These seemingly paradoxical data are consistent with reports that highly functional HSCs have higher mitochondrial mass but exhibit less mitochondrial activity. Thus, DPP4 inhibition may preserve high levels of functional competency by reducing cellular dependence on oxidative phosphorylation. To examine DPP4 inhibition effects on individual subpopulations of HSCs/HPCs, we modeled each cell type separately for differential gene expression. We found that gene programs associated with neutrophil development were significantly enriched in HSCs and MPPs from mice treated with DPA, including genes Elane, Lcn2, Prtn3 and Ctsg/h, concomitant with downregulation of platelet development programs in HSCs and GMPs from mice treated with DPA, including genes Gp6/9, P2ry12 and Clec1b. This suggests that DPP4 drives improved engraftment in part by priming HSCs/HPCs for neutrophil recovery but does not enhance platelet development. We then sought to target a platelet activating pathway in combination with DPP4 inhibition to improve HCT. Recipient mice were treated on days -2, -1, and 0 with 75 mg/kg DPA and day 0 with 50 µg/kg and days 1, 3, 5, and 7 with 25 µg/kg recombinant human Thrombopoietin (TPO), a platelet activating agonist. On day -1 mice were lethally irradiated and transplanted via tail vein with 2.5x10 5 whole BM donor cells on day 0. Complete blood counts 2 weeks following transplantation showed that mice treated with DPA alone exhibited worse platelet recovery, in line with our transcriptomic data. However, mice treated with the combination of DPA and TPO exhibited significantly better platelet recovery than vehicle controls and DPA alone. Further, neutrophil recovery trended towards improved recovery in the combination treatment compared to the single agent treatments. Flow cytometry of the PB revealed no differences in BM homing or in donor PB chimerism one month following HCT in the combination treatment compared to single agents (though all treatments showed improved homing over vehicle controls), suggesting the primary effects were on early recovery and not on initial or long-term engraftment. These data elucidate the DPP4 dependent transcriptome in the hematopoietic compartment, providing molecular insights into genes that drive enhanced functional competency of HSCs/HPCs. We also demonstrate that the DPP4 dependent transcriptome can reveal gene programs that can be targeted in combination to provide additive improvement on transplantation recovery.

In vitro and in vivo killing effects of methionine enkephalin on osteosarcoma

ObjectiveThis study aimed to investigate the underlying regulatory effects of methionine enkephalin (MENK) on osteosarcoma. MethodsThe Cell Counting Kit-8 assay, clone formation, wound healing, transwell assay, and flow cytometry were performed to measure the effects of MENK on the proliferation, migration, invasion, and apoptosis of MG-63 and Saos-2 cells. Opiate growth factor receptor expression (OGFr) in cells was stably knocked down using siRNA. A tumor model was established by inoculating MG-63 cells into mice. Flow cytometry was performed to identify alterations in mice bone marrow, spleen, and tumor tissue immune cells. The phenotype of tumor-associated macrophages was determined using immunohistochemistry. After OGFr knockdown or/and treatment with MENK, Bax, Bcl-2, caspase 3, caspase 9, and PARP expression levels were characterized using qRT-PCR, western blot, and WES, respectively. ResultsMENK could significantly inhibit the proliferation, invasion, and migration of MG-63 and Saos-2, arrest the cell cycle in the G0/G1 phase, upregulate Bax, caspase 3, caspase 9, and PARP expression, and downregulate Bcl-2 expression. Tumor size and weight were lower in the MENK group than those in the control group. MENK-treated mice exhibited a reduced ratio of CD11b + Gr-1 + myeloid-derived suppressor cells. MENK increased the ratio of M1-type macrophages and decreased the proportion of M2-type macrophages in tumor tissue. Furthermore, the level of TNF-α significantly increased while that of IL-10 decreased in MENK-treated mice. The effect of MENK could be partly reversed by OGFr knockdown. ConclusionMENK reduces the abundance of myeloid-derived suppressor cells, induces M1 polarization of macrophages, and exhibits an inhibitory effect on osteosarcoma.

Long non-coding RNA APDC plays important regulatory roles in metabolism of bone and adipose tissues

ABSTRACT The long noncoding RNA (lncR) ANRIL in the human genome is an established genetic risk factor for atherosclerosis, periodontitis, diabetes, and cancer. However, the regulatory role of lncR-ANRIL in bone and adipose tissue metabolism remains unclear. To elucidate the function of lncRNA ANRIL in a mouse model, we investigated its ortholog, AK148321 (referred to as lncR-APDC), located on chr4 of the mouse genome, which is hypothesized to have similar biological functions to ANRIL. We initially revealed that lncR-APDC in mouse bone marrow cells (BMSCs) and lncR-ANRIL in human osteoblasts (hFOBs) are both increased during early osteogenesis. Subsequently, we examined the osteogenesis, adipogenesis, osteoclastogenesis function with lncR-APDC deletion/overexpression cell models. In vivo, we compared the phenotypic differences in bone and adipose tissue between APDC-KO and wild-type mice. Our findings demonstrated that lncR-APDC deficiency impaired osteogenesis while promoting adipogenesis and osteoclastogenesis. Conversely, the overexpression of lncR-APDC stimulated osteogenesis, but impaired adipogenesis and osteoclastogenesis. Furthermore, KDM6B was downregulated with lncR-APDC deficiency and upregulated with overexpression. Through binding-site analysis, we identified miR-99a as a potential target of lncR-APDC. The results suggest that lncR-APDC exerts its osteogenic function via miR-99a/KDM6B/Hox pathways. Additionally, osteoclasto-osteogenic imbalance was mediated by lncR-APDC through MAPK/p38 and TLR4/MyD88 activation. These findings highlight the pivotal role of lncR-APDC as a key regulator in bone and fat tissue metabolism. It shows potential therapeutic for addressing imbalances in osteogenesis, adipogenesis, and osteoclastogenesis.

A Novel Interaction between RUNX1 and the Splicing Factor PTBP1 Is Enriched in Leukemia Cells

RUNX1 and CBFβ form a critical hematopoietic transcription factor complex. RUNX1 is important not only for normal blood development, but also in leukemia. In the context of Inversion 16 acute myeloid leukemia (AML), RUNX1 is required for the activity of CBFβ-SMMHC (CM), the fusion oncoprotein generated by a chromosomal rearrangement and driver of this subtype of AML. The respective roles RUNX1 and CBFβ play in transcriptional regulation are well characterized but the role of other interacting partners is less well understood. To identify novel RUNX1 binding partners, we immunoprecipitated the transcription factor in mouse leukemia cells expressing CBFβ-SMMHC (CM+) and analyzed precipitated proteins by mass spectrometry. One of the proteins identified was the splicing factor Polypyrimidine Tract Binding Protein 1 (PTBP1). PTBP1 is an RNA binding protein that regulates alternative splicing of mRNAs. PTBP1 is required for normal hematopoiesis, including roles in stem cell maintenance, erythroid differentiation, and B-cell selection. However, a role for PTBP1 in leukemia has yet to be described. We confirmed the association of PTBP1 with RUNX1 via immunoprecipitation/western blot in mouse CM+ leukemia cells and the human Inv(16) cell line, ME-1. Next, we asked if this interaction is unique to inv(16) AML. We found that antibodies against RUNX1, but not isotype control antibodies, immunoprecipitated PTBP1 in a variety of myeloid (Molm-13, MV411, Kasumi-1) and lymphoid (Jurkat, MOLT-4, REH, SEM) human leukemia cell lines, indicating that this interaction occurs in multiple leukemia subtypes. To identify the region of RUNX1 required for the interaction with PTBP1, we used two deletion mutants found in patients with RUNX1-Familial Platelet Disorder ( RUNX1-FPD), RUNX1-R232 and RUNX1-Y287. We observed strong binding of PTBP1 with full length RUNX1, but not with either truncation mutant indicating that the RUNX1 C-terminus is required for interaction with PTBP1. To determine location of this novel interaction, we used proximity ligation assay (PLA). We found that the RUNX1:PTBP1 association occurred exclusively in the nucleus in both mouse and human leukemia cells. Next, we asked if this association is observed in healthy hematopoietic cells. By PLA, we found that the RUNX1:PTBP1 interaction is significantly increased in mouse leukemic cells compared to wild type lineage depleted (lin-) mouse bone marrow cells. To determine if the decrease of PLA signal in healthy bone marrow cells was due to spatial separation of PTBP1 and RUNX1, we performed immunofluorescence staining. We observed diffuse nuclear staining of both proteins in the majority of both healthy and leukemic cells. These results indicate that the interaction between RUNX1 and PTBP1 is significantly increased in leukemia cells, despite both proteins being expressed in similar patterns in both healthy and leukemic cells. During our experiments, we noticed large variations in PTBP1 expression levels in different mouse CM+ leukemia samples. To test whether PTBP1 levels correlate with a difference in leukemia stem cell activity, we performed colony assays using equal numbers of leukemia cells from samples with differing PTBP1 expression. We found that samples expressing high levels of PTBP1 resulted in a statistically significant increase in colony number. In addition, the colonies that formed from PTBP1 high samples were notably larger than those formed from PTBP1 low cells, implying a relationship between PTBP1 and leukemia stem cell self-renewal and proliferation. To determine if human AML samples show the same variability in PTBP1 levels, we performed western blot analysis using a panel of nine de novo and relapsed AML samples. Like our findings in mouse leukemia, human samples showed high variability in PTBP1 levels. We are currently addressing whether increased PTBP1 directly promotes leukemia progression. Collectively, our results demonstrate that PTBP1, a splicing regulator, is a novel RUNX1 interacting partner whose association with RUNX1 is significantly increased in leukemia cells. In addition, our data imply a potential role in leukemia stem cell proliferation, self-renewal and a role for PTBP1 in the splicing of RUNX1 target genes, perhaps generating leukemia specific splicing variants. This work has the potential to identify a new component of the RUNX1 transcriptional complex important for leukemogenesis.

Murine Bone Marrow Erythroid Cells Have Two Branches of Differentiation Defined by the Presence of CD45 and a Different Immune Transcriptome Than Fetal Liver Erythroid Cells.

Mouse erythropoiesis is a multifaceted process involving the intricate interplay of proliferation, differentiation, and maturation of erythroid cells, leading to significant changes in their transcriptomic and proteomic profiles. While the immunoregulatory role of murine erythroid cells has been recognized historically, modern investigative techniques have been sparingly applied to decipher their functions. To address this gap, our study sought to comprehensively characterize mouse erythroid cells through contemporary transcriptomic and proteomic approaches. By evaluating CD71 and Ter-119 as sorting markers for murine erythroid cells and employing bulk NanoString transcriptomics, we discerned distinctive gene expression profiles between bone marrow and fetal liver-derived erythroid cells. Additionally, leveraging flow cytometry, we assessed the surface expression of CD44, CD45, CD71, and Ter-119 on normal and phenylhydrazine-induced hemolytic anemia mouse bone marrow and splenic erythroid cells. Key findings emerged: firstly, the utilization of CD71 for cell sorting yielded comparatively impure erythroid cell populations compared to Ter-119; secondly, discernible differences in immunoregulatory molecule expression were evident between erythroid cells from mouse bone marrow and fetal liver; thirdly, two discrete branches of mouse erythropoiesis were identified based on CD45 expression: CD45-negative and CD45-positive, which had been altered differently in response to phenylhydrazine. Our deductions underscore (1) Ter-119's superiority over CD71 as a murine erythroid cell sorting marker, (2) the potential of erythroid cells in murine antimicrobial immunity, and (3) the importance of investigating CD45-positive and CD45-negative murine erythroid cells separately and in further detail in future studies.

Cytogenetic damage by vanadium(IV) and vanadium(III) on the bone marrow of mice

Vanadium is a strategic metal that has many important industrial applications and is generated by the use of burning fossil fuels, which inevitably leads to their release into the environment, mainly in the form of oxides. The wastes generated by their use represent a major health hazard. Furthermore, it has attracted attention because several genotoxicity studies have shown that some vanadium compounds can affect DNA; among the most studied compounds is vanadium pentoxide, but studies in vivo with oxidation states IV and III are scarce and controversial. In this study, the genotoxic and cytotoxic potential of vanadium oxides was investigated in mouse bone marrow cells using structural chromosomal aberration (SCA) and mitotic index (MI) test systems. Three groups were administered vanadium(IV) tetraoxide (V2O4) intraperitoneally at 4.7, 9.4 or 18.8 mg/kg, and three groups were administered vanadium(III) trioxide (V2O3) at 4.22, 8.46 or 16.93 mg/kg body weight. The control group was treated with sterile water, and the positive control group was treated with cadmium(II) chloride (CdCl2). After 24 h, all doses of vanadium compounds increased the percentage of cells with SCA and decreased the MI. Our results demonstrated that under the present experimental conditions and doses, treatment with V2O4 and V2O3 induces chromosomal aberrations and alters cell division in the bone marrow of mice.

Отдаленные последствия γ, n-облучения мышей: снижение длины теломер и развитие опухолей

Purpose: To investigate the telomere length (TL) of bone marrow and thymus cells as a marker of replicative aging late after the prolonged γ, n-irradiation of mice at low and moderate doses and analysis of the appearance of tumors by the end of the experiment − after 14 months. Material and methods: C57Bl/6 and CBA mice were irradiated at doses of 10–500 mGy at the OR-M facility using Pu-Be radionuclide sources at a total absorbed dose rate of neutrons and gamma rays of 2.13 mGy/h, 75 % of which – 1.57 mGy/h – accounted for neutrons with an average energy of 3.5 MeV. Absolute TL in bone marrow and thymus cells was determined using real-time PCR 2 months and 1 year 2 months after irradiation, and the mean TL was calculated. Tumors found during the mice organs examination after autopsy were subjected to histological examination. Results: It was shown that the TL in bone marrow and thymus cells of control CВA mice was 2 times higher than the TL observed in C57Bl/6 mice. Prolonged γ, n-irradiation of C57Bl/6 mice led to a dose-dependent decrease in TL in bone marrow cells 14 months after exposure, which was statistically significant at doses of 100 and 500 mGy. A decreased TL in the thymus was found only at a dose of 500 mGy. During this period, TL in bone marrow cells of CBA mice was reduced in dose-independent manner, starting from as low as 10 mGy, but no statistically significant decrease in TL was found in the thymus. The results obtained indicate the acceleration of replicative senescence of bone marrow cells in mice in the long term period after γ,n-irradiation already at low doses, and in thymus cells only at a dose of 500 mGy. Twenty-four hours after irradiation at doses of 100 and 500 mGy the number of leukocytes in mice of both lines was reduced, which was recovered in C57Bl/6 mice after a week, and in CBA mice – after two weeks. In 14 months after γ, n-irradiation, the appearance of tumors was found in mice of both studied lines: in CBA mice, lung adenocarcinoma at a dose of 50 mGy (in 1 out of 10) and uterine carcinosarcoma at a dose of 500 mGy (in 1 out of 10); in C57Bl/6 mice, keratinizing squamous cell carcinoma of the uterus at a dose of 500 mGy (2 out of 10) was seen in the absence of tumors in control mice. Histological examination of the liver of CBA mice after γ, n-irradiation at a dose of 500 mGy revealed deep dystrophic changes, the causes of which are not clear. Conclusion: The results obtained indicate a high biological hazard of prolonged γ, n-irradiation at doses above 10 mGy, since after irradiation at this dose, an acceleration of replicative senescence of bone marrow cells in the long-term period was found, and the possibility of tumor formation increases after irradiation at a dose of 50 mGy and higher.

DNA recognition and induced genome modification by a hydroxymethyl-γ tail-clamp peptide nucleic acid

Peptide nucleic acids (PNAs) can target and stimulate recombination reactions in genomic DNA. We have reported that γPNA oligomers possessing the diethylene glycol γ-substituent show improved efficacy over unmodified PNAs in stimulating recombination-induced gene modification. However, this structural modification poses a challenge because of the inherent racemization risk in O-alkylation of the precursory serine side chain. To circumvent this risk and improve γPNA accessibility, we explore the utility of γPNA oligomers possessing the hydroxymethyl-γ moiety for gene-editing applications. We demonstrate that a γPNA oligomer possessing the hydroxymethyl modification, despite weaker preorganization, retains the ability to form a hybrid with the double-stranded DNA target of comparable stability and with higher affinity than that of the diethylene glycol-γPNA. When formulated into poly(lactic-co-glycolic acid) nanoparticles, the hydroxymethyl-γPNA stimulates higher frequencies (≥ 1.5-fold) of gene modification than the diethylene glycol γPNA in mouse bone marrow cells.

Evaluation of the Genotoxicity of the Aerial Parts of Iraqi Euphorbia cyathophora on Bone Marrow and Spleen Cells in Mice

The aim of the study was extraction of arial part of Euphorbia cyathophora constituents with methanol and evaluate its effect on mitotic index and total chromosomal aberration bone marrow cell and spleen cell in mice
 200 gm of E. cyathophora fine powder was defatted then extracted by cold maceration 80% ethanol for seven days. The extract was filtered and dried in a rotary evaporator then the dried extract was suspended with water and consecutively extracted using chloroform, ethyl acetate for each. The aqueous layer was then mixed with 100ml methanol. These fractions are dried under reduced pressure to obtain the dry extract. Twenty-four Albino mice were used for the experiment. The animals were divided into four groups: Group 1: Mice were treated with distilled water. The dose was given daily for seven successive days. Group 2: Mice were treated with a single dose (20mg/kg) of methotrexate (positive control). Group 3: Mice were treated with (100mg/kg) of menthol fraction for seven successive days. Group 4: Mice were treated with (200mg/kg) of methanol fraction for seven successive days. Mice were sacrificed by (spinal dislocation). Samples of bone marrow cells and spleen cells were taken and genotoxic analyses.
 Methanol fraction of Euphorbia cyathophora at a dose of 100mg/kg demonstrated a significant decrease in mitotic index and a significant increase in total chromosomal aberrations as compared to distilled water in both bone marrow cells and spleen cells
 (p<0.05). 100 mg/kg and 200 mg/kg of methanol fraction of Euphorbia cyathophora that showed to be significantly higher in mitotic index and significantly lower in total chromosomal aberration as compared to methotrexate (p<0.05).
 In conclusion the current study revealed that the methanol fraction of aerial parts of Euphorbia cyathophora is genotoxic but its genotoxicity is less than that of methotrexate.

Integrated transcriptomic and metabolomic analysis reveals the metabolic programming of GM-CSF- and M-CSF- differentiated mouse macrophages.

Macrophages play a critical role in the inflammatory response and tumor development. Macrophages are primarily divided into pro-inflammatory M1-like and anti-inflammatory M2-like macrophages based on their activation status and functions. In vitro macrophage models could be derived from mouse bone marrow cells stimulated with two types of differentiation factors: GM-CSF (GM-BMDMs) and M-CSF (M-BMDMs), to represent M1- and M2-like macrophages, respectively. Since macrophage differentiation requires coordinated metabolic reprogramming and transcriptional rewiring in order to fulfill their distinct roles, we combined both transcriptome and metabolome analysis, coupled with experimental validation, to gain insight into the metabolic status of GM- and M-BMDMs. The data revealed higher levels of the tricarboxylic acid cycle (TCA cycle), oxidative phosphorylation (OXPHOS), fatty acid oxidation (FAO), and urea and ornithine production from arginine in GM-BMDMs, and a preference for glycolysis, fatty acid storage, bile acid metabolism, and citrulline and nitric oxide (NO) production from arginine in M-BMDMs. Correlation analysis with the proteomic data showed high consistency in the mRNA and protein levels of metabolic genes. Similar results were also obtained when compared to RNA-seq data of human monocyte derived macrophages from the GEO database. Furthermore, canonical macrophage functions such as inflammatory response and phagocytosis were tightly associated with the representative metabolic pathways. In the current study, we identified the core metabolites, metabolic genes, and functional terms of the two distinct mouse macrophage populations. We also distinguished the metabolic influences of the differentiation factors GM-CSF and M-CSF, and wish to provide valuable information for in vitro macrophage studies.

Chemopreventive effect and induction of DNA repair by oenothein B ellagitannin isolated from leaves of Eugenia uniflora in Swiss Webster treated mice

ABSTRACT Oenothein B (OeB) is a dimeric ellagitannin with potent antioxidative, antitumor, immunomodulatory, and anti-inflammatory properties. Despite the promising activities of OeB, studies examining the genotoxic or protective effects of this ellagitannin on DNA are scarce. Therefore, to further comprehensively elucidate the chemopreventive profile of OeB, the aim of this study was to evaluate the mutagenic and antimutagenic actions of OeB using Salmonella typhimurium strains with the Ames test. The micronucleus (MN) test and comet assay were used to assess the anticytotoxic and antigenotoxic effects of OeB on mouse bone marrow cells following differing treatments (pre-, co-, and post-treatment) in response to cyclophosphamide (CPA)-induced DNA damage. In addition, histopathological analyses were performed to assess liver and kidney tissues of Swiss Webster treated mice. Our results did not detect mutagenic or antimutagenic activity attributed to OeB at any concentration in the Ames test. Regarding the MN test, data showed that this ellagitannin exerted antigenotoxic and anticytotoxic effects against CPA-induced DNA damage under all treatment conditions. However, no anticytotoxic action was observed in MN test after pre-treatment with the highest doses of OeB. In addition, OeB demonstrated antigenotoxic effects in the comet assay for all treatments. Histopathological analyses indicated that OeB attenuated the toxic effects of CPA in mouse liver and kidneys. These findings suggest that OeB exerted a chemoprotective effect following pre- and co-treatments and a DNA repair action in post-treatment experiments. Our findings indicate that OeB protects DNA against CPA-induced damaging agents and induces post-damage DNA repair.

Effect of the Anti C-fms Antibody on Osteoclastogenesis in Mouse Bone Marrow Cells In Vitro

Effect of the Anti C-fms Antibody on Osteoclastogenesis in Mouse Bone Marrow Cells In Vitro

Effect of the Anti C-fms Antibody on Osteoclastogenesis in Mouse Bone Marrow Cells In Vitro

Effect of the Anti C-fms Antibody on Osteoclastogenesis in Mouse Bone Marrow Cells In Vitro

Bothrops jararacussu snake venom decreases CD1d, CD83, and CD86 expression on bone marrow-derived dendritic cells

This study was designed to characterize mice bone marrow (BM) and bone marrow-derived dendritic cells (BMDC) and to compare the surface markers expression and inflammatory cytokine liberation in response to LPS and Bothrops jararacussu venom (BjV) stimulation. Typical morphology was observed in BM and BMDCs from the 4th up to the 8th day of culture using recombinant mouse GM-CSF and IL-4. A high basal level of MHC-II, CD1d, CD83, CD11c, CD80, and low CD86 was expressed by BM cells. After stimulation with GM-CSF/IL-4 for BMDCs differentiation, the BM cells differentiated into BMDCs presented MHC-II, CD1d, CD83, CD11c, CD86, and CD80 expression on the 4th – 8th day accompanied with high levels of TNF-α liberated. The difference between the surface markers' expression was observed in this time course in which CD1d, CD11c, and CD80 remained in high levels of expression, while MHC-II and CD83 showed moderate expression during the differentiation period. Also, cytokines liberation was monitored over the period of the BMDCs culture, and on the 6th day, low levels of IL-6 and IL-1β were found, while high levels of TNF-α on the 4th and 8th days, both of which contributed to the maturity of the BMDCs. Maturation of DCs with LPS showed significant upregulation of surface markers (MHC-II, CD1d, CD83, CD86, CD80) and pro-inflammatory cytokines (IL-1β, IL-6, TNF-α) liberation. On the other hand, BjV induced a decrease in CD1d, CD11c, CD83, and CD86 expression in mature BMDCs which was not observed when LPS was used to stimulate BMDCs which probably induces impairment in T-cell activation.

Immunostimulatory effects of marine algae extracts on in vitro antigen-presenting cell activation and in vivo immune cell recruitment.

Marine algae are photosynthetic eukaryotic organisms that are widely used as sources of food, cosmetics, and drugs. However, their biological and immunological effects on immune cells have not been fully elucidated. To unravel their immunological activity and broaden their application, we generated antigen-presenting cells (APCs), including dendritic cells (DCs) and macrophages, from mouse bone marrow cells and treated them with six different marine algae extracts (MAEs). We evaluated cell viability, activation marker expression, and pro-inflammatory cytokine production by APCs after 2 days of MAE treatment. All six MAEs significantly induced cytokine production of APCs, among which Pyropia yezoensis (PY), Peyssonnelia caulifera (PC), and Meristotheca papulosa (MP) extracts exhibited the strongest effect. Cladophora wrightiana var. minor (CW) extract moderately upregulated cytokine levels but increased the expression of activation markers on DCs. Moreover, PY, PC, MP, Sargassum pectinifera (SP), and Caulerpa okamurae (CO) pre-treated APCs effectively stimulated T-cell proliferation and cytokine production. Furthermore, the mice injected with MAEs exhibited higher cytokine (TNF-α, IL-6, and IL-1β) production as well as enhanced innate immune cell recruitment capacities (DCs, monocytes, neutrophils, and natural killer cells) in the peritoneal cavity of the mice compared to those of the non-treated mice. Therefore, all MAEs exhibited immunostimulatory potential, with PY, PC, CW, and MP extracts being the most effective in stimulating immune responses and cell activation. To the best of our knowledge, this is the first study to determine the immunomodulatory activities of six MAEs both in vitro and in vivo.

Concomitant induction of SLIT3 and microRNA-218–2 in macrophages by toll-like receptor 4 activation limits osteoclast commitment

BackgroundToll-like receptor 4 (TLR4) conducts a highly regulated inflammatory process by limiting the extent of inflammation to avoid toxicity and tissue damage, even in bone tissues. Thus, it is plausible that strategies for the maintenance of normal bone-immunity to prevent undesirable bone damage by TLR4 activation can exist, but direct evidence is still lacking.MethodsOsteoclast precursors (OCPs) obtained from WT or Slit3-deficient mice were differentiated into osteoclast (OC) with macrophage colony-stimulating factor (M-CSF), RANK ligand (RANKL) and lipopolysaccharide (LPS) by determining the number of TRAP-positive multinuclear cells (TRAP+ MNCs). To determine the alteration of OCPs population, fluorescence-activated cell sorting (FACS) was conducted in bone marrow cells in mice after LPS injection. The severity of bone loss in LPS injected WT or Slit3-deficient mice was evaluated by micro-CT analysis.ResultWe demonstrate that TLR4 activation by LPS inhibits OC commitment by inducing the concomitant expression of miR-218–2-3p and its host gene, Slit3, in mouse OCPs. TLR4 activation by LPS induced SLIT3 and its receptor ROBO1 in BMMs, and this SLIT3-ROBO1 axis hinders RANKL-induced OC differentiation by switching the protein levels of C/EBP-β isoforms. A deficiency of SLIT3 resulted in increased RANKL-induced OC differentiation, and the elevated expression of OC marker genes including Pu.1, Nfatc1, and Ctsk. Notably, Slit3-deficient mice showed expanded OCP populations in the bone marrow. We also found that miR-218–2 was concomitantly induced with SLIT3 expression after LPS treatment, and that this miRNA directly suppressed Tnfrsf11a (RANK) expression at both gene and protein levels, linking it to a decrease in OC differentiation. An endogenous miR-218–2 block rescued the expression of RANK and subsequent OC formation in LPS-stimulated OCPs. Aligned with these results, SLIT3-deficient mice displayed increased OC formation and reduced bone density after LPS challenge.ConclusionOur findings suggest that the TLR4-dependent concomitant induction of Slit3 and miR-218–2 targets RANK in OCPs to restrain OC commitment, thereby avoiding an uncoordinated loss of bone through inflammatory processes. These observations provide a mechanistic explanation for the role of TLR4 in controlling the commitment phase of OC differentiation.CNR6qWg43XLt4z6Q5VLfxQVideo

Toxicological implications of arecoline N- oxide in vivo in mice test system

Arecoline N- oxide has surfaced as a probable key player of areca nut induced maladies. However, information on the adverse effects of arecoline N- oxide in-vivo is lacking. Mice were divided into 3 groups; the control group was injected with normal saline intraperitoneally while the treatment groups were injected with arecoline N- oxide and arecoline respectively at a dosage of 20 mg/ kg body weight intraperitoneally for thirty days. At the end of the exposure period, end points indicative of toxicity were investigated. Arecoline N- oxide was found to induce DNA damage in mice bone marrow cells. Observable changes in histology of liver indicated arecoline N- oxide to be hepatotoxic. Histological studies also showed arecoline N- oxide to induce infiltration of mononuclear cells in the kidneys. Oxidative stress was induced by arecoline N- oxide in both liver and kidneys as indicated by increased level of lipid peroxidation and decreased level of glutathione, superoxide dismutase and catalase in the tissues. The present study showed arecoline N- oxide to have genotoxic, hepatotoxic and mild nephrotoxic effects in mice which might be due to generation of oxidative stress.