Bone Marrow Cells Research Articles

Identification of the central tolerance checkpoint for autoreactive proteinase 3+ B cells in human bone marrow

Major target antigens of ANCA-associated vasculitis (AAV) are myeloperoxidase (MPO) and proteinase 3 (PR3). High-affinity MPO- and PR3-ANCA immunoglobulins are produced by antigen-experienced, class-switched autoreactive B cells. To prevent autoreactivity, B cells are subjected to several self-tolerance checkpoints, from the early immature stages in the bone marrow (BM), collectively called "central tolerance", to late mature stages, collectively called "peripheral tolerance"; the latter was recently elucidated for autoreactive PR3+ B cells. Here we investigated central tolerance controlling immature PR3+B cells in the BM before their migration into the periphery as transitional B cells. We applied an established flow cytometry-based method using labeled recombinant PR3 (rPR3) to identify the PR3+B cells to compare the phenotype of PR3+B cells in paired samples of BM mononuclear cells (BMMC) and peripheral blood mononuclear cells (PBMC) of non-vasculitis controls (No-AAV), and PBMC of patients with PR3-ANCA-associated vasculitis (PR3-AAV). We observed that the proportion of PR3+B cells within BMMC was higher (median [IQR]; 1.98% [1.77-2.75]) than within PBMC of No-AAV (0.9% [0.63-1.44], p<0.01 by paired comparison) and similar to their proportion within PBMC of patients with PR3-AAV (1.82% [1.66-3.21]; p>0.05). Within CD24++CD38++ B cells, the subset of B cell migrating from BM to the periphery, BMMC contained a greater proportion of PR3+B cells as compared to PBMC in No-AAV (3.35% [1.99-4.92] versus 1.23% [0.62-1.55], p<0.01), showing different surface markers of maturation (i.e. higher proportion of CD27-CD10+ and lower expression of CD21, IgD, IgM). Importantly, we observed a significant decline of the PR3+ fraction from the immature subset (IgD-IgM+; 2.80% [1.23-4.02]) to the early transitional subset (IgD+IgM+; 1.76% [0.96-2.68], p<0.01) in BMMC, while no significant reduction was observed between the early transitional of BMMC and the transitional compartment of PBMC in No-AAV (1.26% [0.62-1.56], p>0.05). In conclusion, to prevent PR3-related autoimmunity, autoreactive PR3+B cells pass a stringent selection in the BM, and their removal by central tolerance checkpoint activity occurs mainly between T1-like/immature to T2-like/early transitional B cells of BMMC.

Influence of glycodelin on the formation of immune response at the level of T-helpers and T-regulatory cells in an &lt;i&gt;in vivo&lt;/i&gt; experiment

Objective. To study the influence of glycodelin on T-helpers and Tregs level in the process of forming an immune response to the introduction of allogeneic bone marrow (BM) cells in a dynamic experiment on Wistar rats. Materials and methods. The original experimental model "host versus transplant reaction" on male Wistar rats without preliminary conditioning in recipients was used in the study. Animals were administered recombinant glycodelin against the background of allogeneic intraperitoneal transplantation of BM cells in a dynamic experiment. The level of peripheral T-helpers (CD4+) and Tregs (CD4+CD25+FOXP3+), and the expression of FOXP3 in the spleen and mesenteric lymph nodes were assessed. The material was collected on the 3rd and 21st days of the experiment. Results. Glycodelin was shown to reduce the absolute number of T-helpers in the peripheral blood (on the 3rd and 21st days) and to increase the proportion of Tregs on the 21st day of the experiment against the background of the introduction of allogeneic BM cells. It was found out, that glycodelin reduced the level of Tregs in the white pulp of the spleen on the 3rd day of the experiment, while the number of these cells on the 21st day increased, reducing the number of T-helpers at the same time. At the level of the mesenteric lymph nodes, glycodelin reduced the level of T-helpers on the 21st day of the experiment, simultaneously increasing the number of Tregs. In general, a unidirectional and distributed effect of glycodelin on the immune response at the level of T-helpers was observed, that was a decrease of T-helpers, but an increase of Tregs on the 21st day of the experiment. Conclusion. Thus, glycodelin had an immunomodulatory effect on T-helpers and Tregs formation. The vector of the obtained effects was immunosuppressive in nature and contributed to the suppression of the immune response to allogeneic cells.

Inflammatory crosstalk impairs phagocytic receptors and aggravates atherosclerosis in clonal hematopoiesis in mice.

Clonal hematopoiesis (CH) increases inflammasome-linked atherosclerosis but the mechanisms by which CH mutant cells transmit inflammatory signals to non-mutant cells are largely unknown. To address this question we transplanted 1.5% Jak2VF bone marrow (BM) cells with 98.5% WT BM cells into hyperlipidemic Ldlr-/- mice. Low allele burden (LAB) mice showed accelerated atherosclerosis with increased features of plaque instability, decreased levels of macrophage phagocytic receptors MERTK and TREM2, and increased neutrophil extracellular traps (NETs). These changes were reversed when Jak2VF BM was transplanted with Il1r1-/- BM. LAB mice with non-cleavable MERTK in WT BM showed improvements in necrotic core and fibrous cap formation and reduced NETs. An agonistic TREM2 antibody (4D9) markedly increased fibrous caps in both control and LAB mice eliminating the difference between groups. Mechanistically, 4D9 increased TREM2+PDGFB+ macrophages and PDGF receptor-α positive fibroblast-like cells in the cap region. TREM2 and PDGFB mRNA levels were positively correlated in human carotid plaques and co-expressed in macrophages. In summary, low frequency Jak2VF mutations promote atherosclerosis via IL-1 signaling from Jak2VF to WT macrophages and neutrophils promoting cleavage of phagocytic receptors and features of plaque instability. Therapeutic approaches that stabilize MERTK or TREM2 could promote plaque stabilization especially in CH- and inflammasome-driven atherosclerosis.

Abstract 4137935: Adipogenesis in Bone Marrow Niche under Cardiac Stress Worsens Cardiac Function

Background: We have recently reported that repetitive cardiac decompensation with multimorbidity often experienced by patients with heart failure (HF) is attributed to epigenetic modifications of hematopoietic stem cells (HSCs) in the bone marrow (BM). HF reprogrammed HSCs differentiation and altered tissue macrophage homeostasis. These findings demonstrate that the BM can carry an innate immune memory of cardiac stress that may exacerbate HF and predispose other organs to pathology. Aims: Because the stemness of HSCs is mainly regulated by mesenchymal stromal cells (MSCs) in the BM niche, we investigated phenotypic alterations of MSCs under cardiac stress. Methods&amp;Results: Transcriptome analysis of MSCs showed preferential differentiation toward adipocytes in murine pressure overload models. In vitro assays and histological BM sections also support this finding. Furthermore, single-cell RNA sequencing of MSCs demonstrated that the percentage of adipocyte-primed MSCs increased in proportion to the severity of cardiac dysfunction, and also correlated with the frequency of myeloid-lineage progenitor cells. To investigate the influence of adipo-lineage MSCs on HSCs, we conducted BM transplantation supplemented with MSCs from control or HF mice. Recipient mice transplanted with HF-MSCs showed significant increases in myeloid-biased multipotent progenitors in BM and myeloid cells in peripheral blood. Additionally, the number of proinflammatory cardiac macrophages was significantly increased in the HF-MSCs group, promoting cardiac fibrosis and dysfunction. Conclusions: Our results demonstrated that the BM niche could perceive cardiac stress in the form of adipocytic skewing of MSCs in the setting of HF, which changed the differentiation behavior in HSCs and ultimately led to further deterioration of cardiac function through the impaired differentiation of circulating monocytes into cardiac macrophages. Therefore, suppressing the adipocytic differentiation of MSCs could have a novel therapeutic potential to avoid repeated HF events.

Abstract 4130641: The Role of Oxidized Phospholipids in Triggering Trained Immunity and Accelerating Atherosclerotic Plaque Inflammation during Cholesterol Cycling

Introduction: Oxidized phospholipids (OxPL) derived from LDL play a crucial role in the development and progression of atherosclerosis by promoting chronic inflammation and formation of foam cells. The natural antibody E06, binding to OxPL, diminishes macrophage uptake of OxLDL in vitro and decreases atherosclerosis progression in vivo. In our prior work using a model of plasma cholesterol cycling (high-low-high), resulting in atherosclerotic plaque Progression (P)-Regression (PR)-re-Progression (PRP), respectively, we observed accelerated plaque inflammation in the PRP group relative to mice with non-cycling high cholesterol. Hypothesis: OxPL promotes plaque inflammation in the cholesterol cycling model via trained immunity and that the E06 antibody will mitigate this process. Methods: We utilized transgenic "E06 mice" that expressed a single-chain variable fragment of E06 (E06-scFv) using the Apoe promoter on the Ldlr−/− background. Both E06 mice and Ldlr-/- mice were subjected to high cholesterol diets (HCD) and categorized into three groups in the cholesterol cycling model: Baseline Progression (P), PR and PRP. Plaque regression was induced by ApoB-ASO injection, which significantly lowered cholesterol levels by inhibiting LDL production. Results: As before, Ldlr-/- mice in the PRP group exhibited accelerated plaque inflammation post-regression shown by CD68 staining on aortic roots. Notably, this acceleration was significantly inhibited in the E06 mice, highlighting the protective effects of E06 against plaque re-progression. Additionally, flow cytometry of peripheral blood exhibited reduced total monocytes and Ly6Chi-monocytes (thought to become inflammatory macrophages in plaques), alongside increased Ly6Clo-monocytes (thought to become inflammation resolving macrophages in plaques) among E06 mice in the PRP group compared to Ldlr-/- mice. Ex-vivo stimulation of bone marrow cells with the inflammatory stimulus LPS demonstrated higher IL-6 levels in supernatant in Ldlr-/- mice versus E06 mice in the PRP group, suggesting OXPL-induced trained immunity in bone marrow precursors of monocytes and macrophages is involved. Conclusion: The acceleration of plaque inflammation in cholesterol cycling model was significantly inhibited in the E06 mice. These results provide insight into the increased risk of cardiovascular disease in individuals with intermittent adherence to statins who undergo cholesterol cycling.

DDDR-34. A SYSTEMS MEDICINE PLATFORM FOR INDIVIDUALIZED TREATMENT OF GLIOBLASTOMA

Abstract Glioblastoma is an aggressive brain cancer characterized by complex intertumoral heterogeneity. Yet, no methods have successfully stratified patients for effective individualized treatments. We aimed to use a systems medicine approach to individualize treatments for patients. From 27 patients undergoing surgery for either a newly diagnosed or a progressive glioblastoma, we collected biopsies and cultured the cells as spheroids. Drug sensitivity testing for &amp;gt;500 drugs from the FDA-approved oncology collection was performed. Drug efficacy was quantified using a modified area under the dose-response curve (Drug Sensitivity Score, DSS), and individualized by normalizing the DSS to the drug response in bone marrow cells from healthy donors (selective DSS, sDSS). All samples were assayed by WES, WGS, and RNA-sequencing. We found that glioblastomas can be stratified into three DSS groups. These were associated with expression of genes both known (e.g. ALDH1A1), and not known (e.g. VRK2) to be linked to drug resistance. By unsupervised clustering of sDSS, we found a robust patient stratification primarily driven by drug sensitivity to four groups of targets: i) EGFR, ii) FGFR/PDGFR/VEGFR, iii) MDM2 and iv) MEK/ERK. We found differentially expressed genes (N = 20-90 genes, varying by group) between the responders and non-responders in each group. We explored differential pathway activity between the responders and non-responders in each group to correlate drug responses to transcriptional networks to inform underlying disease mechanisms. The transcriptional networks further provide a molecular basis for drug predictions for additional treatment prioritizations. We conclude that our platform can stratify glioblastomas into subgroups, inform the underlying biology of drug sensitivity, and prioritize treatments for patients. Clinical utility is currently under evaluation in a formal trial.

IMMU-15. CD74/MIF SIGNALING AXIS DISRUPTION IN BONE MARROW-DERIVED MYELOID CELLS DELAYS MALIGNANT PROGRESSION IN A PRECLINICAL MODEL OF GLIOMA

Abstract INTRODUCTION CD74, a chaperone associated with the major histocompatibility complex class II (MHC-II), plays a crucial role in assembling and transporting MHC-II molecules on the cellular surface. Beyond its physiological function in antigen presentation, CD74 is a negative regulator of inflammation, and its dysregulation is associated with inflammatory disorders and cancer. Notably, tumor stem cells release macrophage migration inhibitory factor (MIF) to trigger CD74 immunosuppressive signaling in tumor-infiltrating immune cells, thereby abrogating CD8+ T cell activation and promoting angiogenesis. While CD74 expression is known in dendritic cells, macrophages and B cells, our recent findings reveal its upregulation in immunosuppressive macrophages within the low-grade glioma (LGG) tumor microenvironment, and its negative correlation with the survival of adolescent and young adult patients with LGG. HYPOTHESIS. We hypothesize that inhibiting CD74/MIF in immunosuppressive myeloid cells may affect tumor progression and prevent malignant transformation (MT) to high-grade glioma (HGG). METHODS To test our hypothesis, we isolated bone marrow cells from immunocompetent RCAS n/tv-a LGG bearing animals, differentiated them into bone marrow-derived myeloid cells (BMDMs), silenced CD74 expression, and co-cultured them with primary CD8+T cells. Moreover, we inhibited CD74/MIF signaling in vivo. RESULTS. Deconvolution analysis of tumor-infiltrating immune cells revealed upregulation of CD74 and its network in myeloid cells during LGG compared to HGG. Silencing CD74 in LGG BMDMs in vitro resulted in the downregulation of genes associated with migration, proliferation, and immunosuppression. The co-culture of LGG CD74KD-BMDMs with CD8+ T cells increased the expression of T cell activation genes. In vivo treatment of LGG mice with MIF inhibitors significantly decreased myeloid cell infiltration in the tumor microenvironment (TME) and prolonged survival. CONCLUSIONS These preclinical studies highlight a role for the CD74/MIF signaling axis in glioma and present a novel immunotherapeutic target to modulate the activity of immunosuppressive myeloid cells in order to delay MT.

Protective effect of bone-marrow stromal cells on injured cortical neurons subjected to hemostimulation in vitro

Objective: Bone-marrow mesenchymal stem cells (BMSCs) are a subpopulation of cells found in the bone marrow stromal of mammals that have the potential to differentiate and form bone, cartilage, adipose, neural, and muscle cells, with strong proliferative ability, multidirectional differentiation potential, immunomodulatory function. Here, we reported the novel findings on the effect of BMSC in protecting injured cortical neurons induced by hemostimulation. Methods: Cortical neurons harvested from the neonatal rat and were isolated and incubated at 37°C in a 5% CO2 environment. During this process, part of cells was subjected to hemostimulation, and BMSC derived supernatant addition was performed to observe its effect on neuroprotection. Hoechst 33342/PI co-staining and CCK-8 cell viability assays were utilized to evaluate the effects of BMSC supernatant administration on primary cortical neurons. Results: BMSC derived supernatant administration effectively protects hemoglobin-induced neuronal damage, indicating by cell viability-detection. In addition, BMSCs exert optimal effect by inhibiting neuronal cell apoptosis. Conclusions: BMSC derived supernatant treatment effectively ameliorates hemoglobin-induced neuronal damage and attenuates neuronal apoptosis.

Comparative analysis of whole-genome sequences of African swine fever virus (Asfarviridae: Asfivirus) isolates сollected on the territory of the left bank of the Dnieper River in 2023.

The lack of data on the whole-genome sequences of African swine fever virus (ASFV) variants circulating on the territory of the left bank of the Dnieper River complicates the understanding of the molecular evolution of the virus and the character of the epidemic process development in Russia and Ukraine. Understanding the genetic divergence and phylogenetic relatedness of isolates can largely adjust the strategy of general and specific prevention of the disease. The aim of the study - search and description of unique mutations (deletions/insertions/substitutions) in isolates collected from domestic pigs in Donetsk, Luhansk and Zaporozhye regions in 2023; determination of relatedness and level of homology with reference strains of ASFV genotype II; sub-genotyping and clustering of isolates based on whole-genome analysis. The samples used were a culture suspension of porcine bone marrow (PBM) cells containing ASFV isolates obtained from pathologic material from domestic pig carcasses. Genomic DNA was prepared by purification and concentration of virus followed by phenol-chloroform extraction of total nucleic acid. The high-throughput sequencing process was performed using MGI technology. Consensus sequences were assembled by mapping reads to the reference genome of strain Georgia 2007/1. All isolates are assigned to genotype II, have a monophyletic origin, are phylogenetically close to the clusters «Europe» (4/5) and «Bryansk 2021» (1/5), and are divergent from the original parental genetic variants that make up the enlarged clades. In addition, numerous substitutions in the loci of the multigene family MGF 110, 505, and 360, encoding virulence proteins, were detected in 4 isolates from Donetsk and Zaporozhye regions. The phylogeny of the genotype II ASFV, which originated from the reference strain Georgia 2007/1, is shown to be sufficient for isolate differentiation. The presented data are of theoretical and practical importance for domestic and international ASFV surveillance.

Bone marrow versus peripheral blood allogeneic haematopoietic stem cell transplantation for haematological malignancies in adults.

Allogeneic haematopoietic stem cell transplantation (allo-HSCT) is an established treatment option for many malignant and non-malignant haematological disorders. Peripheral blood stem cells represent the main stem cell source in malignant diseases due to faster engraftment and practicability issues compared with bone marrow stem cells. Since the early 2000s, there have been many developments in the clinical field. Allo-HSCT using haploidentical family donors (haplo-HSCT) has emerged as an alternative for people who do not have human leukocyte antigen (HLA)-matched siblings or unrelated donors. In addition, the introduction of new methods and strategies in allo-HSCT, such as the use of post-transplant cyclophosphamide (PT-Cy), better donor selection, the more frequent administration of anti-thymocyte globulins (ATGs), but also improved management of side effects such as graft-versus-host disease (GvHD) and infection, have impacted outcomes after allo-HSCT. In addition, as transplant indications and strategies continue to adapt in line with novel research findings, the effect of the stem cell source on post-transplant outcomes is unclear. For our analysis, we considered peripheral blood stem cells as the standard graft source for adults with haematological malignancies. This is an update of a review first published in 2014. To assess the effect of bone marrow transplantation versus peripheral blood stem cell transplantation in adults with haematological malignancies with regard to overall survival, disease-free survival, incidence of non-relapse or transplant-related mortality, incidence of extensive chronic graft-versus-host disease (GvHD), incidence of acute GvHD grades III to IV, incidence of overall chronic GvHD, and quality of life. For this update we searched CENTRAL, MEDLINE, Embase, and two trials registries on 2 November 2022 with no language restrictions. We included randomised controlled trials (RCTs) comparing bone marrow transplantation (BMT) with peripheral blood stem cell transplantation (PBSCT) in adults (aged ≥ 18 years) with haematological malignancies. Two review authors independently selected studies and extracted data. We evaluated risk of bias using the original Cochrane risk of bias tool (RoB 1), and we evaluated the certainty of the evidence using the GRADE approach. The updated search identified no new studies for inclusion. We found two additional reports relating to a previously included study; they provided new data on quality of life and infection rates after transplantation. As these are clinically relevant outcomes, quality of life was added to the summary of findings table (replacing acute GvHD II to IV), and rate of infection was added to our list of secondary outcomes. We included nine RCTs with a total of 1521 participants. Overall, the risk of bias in the included studies was low. Median participant age across studies ranged from 21 to 45 years, and studies took place in Canada, the USA, New Zealand, Brazil, Australia, Egypt, and across Europe. Bone marrow transplantation (BMT) compared with peripheral blood stem cell transplantation (PBSCT) likely results in little to no difference in overall survival (hazard ratio (HR) for all-cause death 1.07, 95% CI 0.91 to 1.25; 6 studies, 1330 participants; moderate-certainty evidence). There may be little to no difference between BMT and PBSCT in terms of disease-free survival (HR for disease recurrence or all-cause death 1.04, 95% CI 0.89 to 1.21; 6 studies, 1225 participants; low-certainty evidence) and non-relapse or transplant-related mortality (HR 0.98, 95% CI 0.76 to 1.28; 3 studies, 758 participants; low-certainty evidence). BMT compared with PBSCT likely results in lower rates of extensive chronic GvHD (HR 0.69, 95% CI 0.54 to 0.90; 4 studies, 765 participants; moderate-certainty evidence) and overall chronic GvHD (HR 0.72, 95% CI 0.61 to 0.85; 4 studies, 1121 participants; moderate-certainty evidence). BMT compared with PBSCT may reduce the incidence of acute GvHD grades III to IV, although the 95% CI of the HR is also compatible with no effect (HR 0.75, 95% CI 0.55 to 1.02; 3 studies, 925 participants; moderate-certainty evidence). Evidence from two trials that used different quality of life assessment instruments suggests that BMT compared with PBSCT may be associated with higher quality of life five years after transplantation. Moderate-certainty evidence suggests little to no difference in overall survival following allo-HSCT using bone marrow versus peripheral blood stem cells (the current clinical standard stem cell source). Low-certainty evidence suggests little to no difference between the stem cell sources in terms of disease-free survival and non-relapse or transplant-related survival. BMT likely reduces the risk of extensive chronic GvHD and overall chronic GvHD compared with PBSCT. Evidence from two RCTs suggests that BMT compared with PBSCT may result in higher long-term quality of life, possibly due to the lower chronic GvHD incidence. With this update, we aimed to supply the most recent data on the choice of stem cell source for allo-HSCT in adults by including new evidence published up to November 2022. We identified no new ongoing studies and no new RCTs with published results. Further research in this field is warranted.

YTHDC1 is a therapeutic target for B-cell acute lymphoblastic leukemia by attenuating DNA damage response through the KMT2C-H3K4me1/me3 epigenetic axis.

B-cell acute lymphoblastic leukemia (B-ALL) is an aggressive malignancy characterized by the aberrant accumulation of immature and dysfunctional B cells in bone marrow (BM). Although chemotherapy and other therapies have been widely applied, some patients such as relapsed ordrug-refractory (R/R) B-ALL patients exhibit limited response. YT521-B homologous domain-containing protein 1 (YTHDC1) is a nuclear reader of N6-methyladenosine (m6A) RNA modification, which has been implicated in different malignancies including leukemia. In the current study, we show that YTHDC1 is highly expressed in B-ALL cells. YTHDC1 knockdown attenuated B-ALL cell proliferation and cell cycle progression in vitro, and prolonged survival of mice in the human B-ALL xenograft model in vivo attributable to compromised leukemogenesis. Mechanistically, YTHDC1 knockdown significantly increased the accumulation of endogenous and chemotherapeutic agents-induced DNA damage in B-ALL cells. Furthermore, we identified that YTHDC1 binds to and stabilizes m6A-modified KMT2C mRNA. KMT2C is a key enzyme catalyzing histone H3K4 methylation required for the expression of DNA damage response (DDR)-related genes, implying that YTHDC1 inhibitors might improve chemotherapy by attenuating DDR via reducing KMT2C. Indeed, with molecular docking and biochemical experiments, we identified EPZ-5676 as a YTHDC1 inhibitor, and combination of EPZ-5676 with Cytarabine (Ara-c) significantly improved the efficacy of chemotherapy in B-ALL mouse models using YTHDC1high primary and lined B-ALL cells. Collectively, YTHDC1 is required for DDR in B-ALL cells by upregulating DDR-related gene expression via stabilizing m6A-modified KMT2C mRNA, thereby leading to increased histone H3K4 methylation, and targeted inhibition of YTHDC1 is a potentially new therapeutic strategy against B-ALL, especially YTHDC1high B-ALL.

F-Box and WD repeat domain containing 7 induces infectious osteomyelitis by regulating MYB stability and ubiquitination.

Osteomyelitis is a bone inflammation initiated by invading pathogens. Macrophages and inflammation play essential roles in osteomyelitis. F-Box and WD repeat domain containing 7 (Fbxw7) is a tumour suppressor and E3 ubiquitin ligase. In the present study, the potential roles of Fbxw7 in osteomyelitis were explored. The mRNA level of Fbxw7 was measured in bone marrow cells from patients with osteomyelitis and Staphylococcus aureus (S. aureus)-infected macrophages. The conditional knockout mice with Fbxw7 deficiency in myeloid cells were generated. The expression of interleukin (IL)-6, IL-23a and nitric oxide synthase 2 (Nos2) was measured in S. aureus-infected Fbxw7-deficient bone marrow-derived macrophages (BMDMs). The body weight loss, bacterial burden, bone loss and formation and serum level of IL-6, IL-23 and TNF-α were measured in S. aureus-infected Fbxw7 conditional KO mice. The interacting partners of Fbxw7 were predicted using STRING and the interaction were tested. Elevated expression of Fbxw7 was observed in bone marrow cells from patients with osteomyelitis and in S. aureus-infected macrophages. The expression of IL-6, IL-23a and Nos2 was remarkably suppressed in S. aureus-infected Fbxw7-deficient BMDMs. Fbxw7 conditional knockout mice had less body weight loss, higher bacterial burden, less bone loss and formation and decreased serum level of cytokines. Fbxw7 interacted with MYB. S. aureus-infected Fbxw7-deficient BMDMs had higher level of MYB and less ubiquitination of MYB. Fbxw7 promotes osteomyelitis symptoms by regulating ubiquitination and stability of MYB.

Identification of CD141+vasculogenic precursor cells from human bone marrow and their endothelial engagement in the arteriogenesis by co-transplantation with mesenchymal stem cells

BackgroundCritical limb ischemia (CLI) is a condition characterized by insufficient blood flow to the lower limbs, resulting in severe ischemia and potentially leading to amputation. This study aims to identify novel vasculogenic precursor cells (VPCs) in human bone marrow and evaluate their efficacy in combination with bone marrow-derived mesenchymal stem cells (BM-MSCs) for the treatment of CLI.MethodsEx vivo cultured VPCs and BM-MSCs from bone marrow were characterized and their effects on neovascularization and long-term tissue regeneration were tested in a mouse CLI model.ResultsVPCs, expressing high levels of hepatocyte growth factor and c-MET, were identified from human bone marrow aspirates. These cells exhibited strong vasculogenic capacity in vitro but possessed a cellular phenotype distinct from those of previously reported endothelial precursor cells in circulation or cord blood. They also expressed most surface markers of BM-MSCs and demonstrated multipotent differentiation ability. Screening of 376 surface markers revealed that VPCs uniquely display CD141 (thrombomodulin). CD141+VPCs are present in BM aspirates as a rare population and can be expanded ex vivo with a population doubling time of approximately 20 h, generating an elaborate vascular network even under angiogenic factor-deficient conditions and recruiting BM-MSCs to the network as pericyte-like cells. Intramuscular transplantation of a combination of human CD141+VPCs and BM-MSCs at a ratio of 2:1 resulted in limb salvage, blood flow recovery, and regeneration of large vessels in the femoral artery-removed CLI model, with an efficacy superior to that of singular transplantation. Importantly, large arteries and arterioles in dual cell transplantation expressed human CD31 in the intima and human α-smooth muscle actin in media layer at 4 and 12 weeks, likely indicating their lineage commitment to endothelial cells and vascular smooth muscle, respectively, in vivo.ConclusionDual-cell therapy using BM-derived CD141+ VPCs and BM-MSCs holds potential for further development in clinical trials to treat peripheral artery disease and diabetic ulcers.

Bilateral exudative retinal detachment with subretinal light-chain protein in a patient with multiple myeloma -case report-

BackgroundExudative retinal detachment in patients with multiple myeloma is exceedingly rare. Only two Cases are known to us. We successfully identified light-chain proteins in subretinal fluid, allowing for a more precise understanding of the pathogenesis of this complication.Case presentationA 68-year-old patient presented with bilateral exudative retinal detachment. The visual impairment was reported one day after stem cell mobilization by granulocyte-colony stimulating factor (G-CSF) and the additional administration of Plerixafor. The symptoms began during stem cell apheresis. The patient underwent surgical treatment for both eyes through vitrectomy and silicone oil tamponade. Light-chain proteins were detected in the collected subretinal fluid through electrophoresis in one eye.ConclusionsWe successfully identified light-chain proteins in subretinal fluid, allowing for a more precise understanding of the pathogenesis of this complication. The pathomechanism likely involves damage to the outer blood-retina barrier due to the deposition of light-chain proteins. Whether mobilization of bone marrow cells with Plerixafor led to a breakdown of the outer blood-retina barrier in these patients is a topic for discussion and has to be considered in the use of Plerixafor.

DDIT4 promotes erythroid differentiation and coordinates with SIPA1 to regulate erythroid proliferation in bone marrow of high altitude erythrocytosis

Erythrocytosis moderately enhances the oxygen-carrying capacity of the blood and is considered a characteristic response of individuals adapting from low-altitude regions to high-altitude regions. Nevertheless, erythrocytosis can also turn excessive and result in maladaptive syndromes, such as high altitude polycythemia (HAPC). The increased differentiation or proliferation of erythroid cells in the bone marrow may be a crucial factor leading to accumulation of peripheral erythroid cells. However, the mechanism of erythroid regulation within the bone marrow of high-altitude erythrocytosis remains insufficiently systematically observed. We utilized single-cell transcription sequencing to characterize bone marrow cells following chronic hypoxic exposure and found that bone marrow erythrocytosis is associated with the accumulation of Baso-E, Poly-E, and Ortho-E cells at the terminal stage of erythroid lineage differentiation. Through analysis of differential gene expression and localization in differentiated cells within the erythroid lineage, we confirmed that DDIT4 expression was localized in advanced differentiated erythroblast including Baso-E, Poly-E and Ortho-E, its expression was significantly enhanced by hypoxia exposure. We demonstrated that overexpression of DDIT4 could promote K562 cell differentiation, and through the IP pull-down interaction protein profile, we found that DDIT4 might participate in regulating the cell cycle by interacting with SIPA1 to promote the proliferation of erythroid cells and may be involved in HAPC.

FLAER Revealed Normally Expected Non-PNH FLAER-Dim Immature Myeloid Cells (CD117+/CD34-) In Bone Marrow Aspirates and Could Be Utilized as a Marker of Hierarchical Hematopoiesis.

Fluorescently labeled aerolysin (FLAER) is widely used for the identification of paroxysmal nocturnal hemoglobinuria (PNH) clones in peripheral blood (PB) samples. However, there are only a few reports on the differential fluorescent intensity of FLAER in normal bone marrow (BM) cell subpopulations. The purpose of this study was to evaluate FLAER expression during normal and pathological hematopoiesis, to map the critical existence of non-PNH FLAER-dim cells. A total of 54 BM aspirates were prospectively analyzed with FLAER-based flow cytometric (FC) protocols, during their routine work-up. These were obtained from patients with the following diagnoses: PNH (3), infections/reactive (5), myelodysplastic syndromes (MDS) (7), myelodysplastic/myeloproliferative neoplasms (MDS/MPN) (4), chronic myelogenous leukemia (CML) (3), acute myelogenous leukemia (AML) at diagnosis (20), AML in measurable residual disease (MRD) assessment (7), and B-cell acute lymphoblastic leukemia (B-ALL) in MRD assessment (5). The applied protocols consisted of FLAER, HLA-DR, CD14, CD33, CD34, CD66b, CD38, CD117, CD64, CD45, and FLAER, CD66c, CD14, CD33, CD34, CD66b, CD123, CD16, CD64, and CD45, respectively. FLAER expression was assessed in CD34++/CD38- and CD34+/CD38+ stem cells, CD34-/CD117+/HLA-DR+/CD33+ myeloid precursors, and CD64+/CD14-/HLA-DR+ monocyte precursors but also in mature myeloid cells. All patients revealed an intermediate FLAER intensity in CD34++/CD38- stem cells, with a discrete FLAER-negative subpopulation observed only in maturing CD34+/CD38+ stem cells of patients with PNH. The lowest FLAER intensity was noticed in CD34-/CD117+/HLA-DR+/CD33+ myeloid precursors, not only in patients with PNH but also in PNH-negative BM aspirates. An ascending FLAER intensity was further observed during monocytic and granulocytic maturation, with a discrete FLAER-negative population in CD64+/CD14-/HLA-DR+ monocyte precursors and maturing neutrophils and monocytes of patients with PNH only. The maturation pattern of FLAER expression was further confirmed in a patient with acute promyelocytic leukemia treated with all-trans retinoic acid (ATRA), where FLAER was concurrently upregulated with CD66b in a consecutive series of PB samples tested over a 20-day-period after diagnosis. The application of FLAER in PNH-positive and PNH-negative reactive or malignant BM aspirates identified normally expected non-PNH FLAER-dim CD34-/CD117+/HLA-DR+/CD33+ myeloid precursors in all samples. A specific FLAER-associated maturation pattern was observed, which is proposed for further study within MRD and diagnostic protocols.

Sterility of cord blood units: Evaluation of the collection, preparation and conservation process with a view to establishing a bank of stem cells from cord blood at the Biological Resources Center of the Institut Pasteur de Côte d’Ivoire (2024)

Introduction: Cord blood stem cells are used as an alternative to bone marrow and peripheral blood cell transplantation. For this purpose, cord blood banks have been established worldwide for the collection and cryopreservation of cord blood units. These banks must implement rigorous protocols to ensure the microbiological safety of grafts. The aim of this study is to evaluate the process of selecting mothers and collecting and processing these samples developed for this purpose. Methodology: Pregnant women were enrolled in the study on the basis of selection criteria. Maternal blood and cord blood samples were collected by the in utero method. Serological analyses of maternal blood for the detection of vertically transmitted infections and microbiological examinations of cord blood units were carried out. Results: 31 women out of 46 (68.4%) were enrolled. All had negative serology for the targeted pathogens. Similarly, microbiology revealed no microbial contamination of the cord blood units. Conclusion: This work demonstrates that the procedures for selecting mothers and processing implementations are effective in ensuring the microbiological safety of cord blood units and could be suggested in the context of banking cord blood stem cells at the Institut Pasteur de Côte d’Ivoire.

Granulocyte colony-stimulating factor for stem cell mobilisation in acute myocardial infarction: a randomised controlled trial

BackgroundTo determine whether granulocyte colony-stimulating factor (G-CSF) improves clinical outcomes after large ST-elevation myocardial infarction (STEMI) when administered early in patients with left ventricular (LV) dysfunction after successful percutaneous coronary...

Exposure to elevated cholesterol results in a non-reversible epigenetic and metabolic shift in macrophages associated with increased residual inflammatory risk

Abstract Almost all patients with or at high risk of atherosclerosis are treated with statin therapy. However, there remains a high burden of cardiovascular risk in these patients. Indeed, it is now clear that residual inflammatory risk is a stronger predictor of cardiovascular events, cardiovascular death, and all-cause death than residual cholesterol risk (as measured by low-density lipoprotein cholesterol). Therefore, in this study we sought to better understand the origin of inflammatory residual risk associated with previous exposure to high cholesterol. Tissue resident macrophages and bone marrow derived macrophages (BMDM) from mice which have been exposed to high cholesterol display an augmented polarisation profile. BMDM’s generated ex vivo are grown under standard condition so any effects come from in vivo exposure to high cholesterol. BMDMs from high cholesterol mice display a heighten M1 and blunted M2-like phenotype. Metabolomic analysis of M(IL-4) macrophages reveal they have breaks in the TCA cycle resulting in reduced capacity to utilise OxPhos which is needed for full M(IL-4) polarisation. Macrophages from chimeric mice generated by bone marrow transfer of high cholesterol bone marrow into irradiated normo-cholesterol mice retain the same phenotype as macrophages from high-cholesterol mice. Critically we demonstrate that there are genome wide irreversible epigenetic changes in bone marrow cells and tissue resident macrophages as a result of exposure to high cholesterol. Mice from a high cholesterol background have an increased number of total HSC (lin-ckit+sca1+) within the bone marrow, however, there is a decreased in the total number of long term (LT)-HSC; critically, this phenotype is not reversible when lipids levels are normalised using monoclonal antibody therapy against PCSK9. This is coupled with an irreversible myeloid skewing in the bone marrow immune cell composition. Newly recruited macrophages within the adipose also retain a heighten M1 and blunted M2-like phenotype despite lipid normalisation conferring a negative systemic metabolic phenotype. Critically, these data show that cholesterol induces long term epigenetic and metabolic changes to HSC and macrophages, which may account for residual inflammatory risk.

DNMT3A CHIP-driver mutations ignite the bone marrow

Abstract Background Clonal hematopoiesis of indeterminate potential (CHIP) is an acquired, genetic cardiovascular (CV) risk factor affecting about 10% of the general population at the age of 70 years. Most CHIP-driver mutations affect the epigenetic regulator gene DNMT3A. Experimental studies suggest a causal link between CHIP and inflammation-driven atherosclerosis, but the underlying mechanisms of how few mutant clones provoke adverse clinical outcomes in patients remain obscure. Methods Single monocytes and bone marrow cells from DNMT3A-mutation carriers undergoing coronary angiography and open-heart surgery underwent RNA and genomic DNA sequencing in parallel on a single cell level. This approach enabled us to compare mutant and non-mutant cells directly within carriers, and to non-carrier cells. Atherosclerotic chimeric mice reconstituted with a mixture of Dnmt3a-deficient (CD45.2) and -competent (CD45.1) bone marrow served as an experimental model to compare mutant to non-mutant macrophages in atherosclerotic lesions. Results Surprisingly, RNA/gDNA parallel sequencing revealed that gene expression profiles of mutant monocytes resembled those of non-mutant monocytes in carriers of DNMT3A-CHIP mutations (n=4). Collectively, however, monocytes of DNMT3A mutation carriers were more inflammatory than those of matched non-carrier patients. Mutant hematopoietic stem and progenitor cells, analyzed by RNA/gDNA parallel sequencing in two carriers, expressed more activation and inflammatory markers compared to non-mutant cells within the same bone marrow, and intraindividual differences declined as cells differentiated into monocytes. Macrophages were isolated from atherosclerotic aortas of mixed bone marrow chimeric mice based on CD45.1 (non-mutant) or CD45.2 (mutant or non-mutant controls) expression and subjected to single cell RNA sequencing. Inflammation markers were higher expressed in non-mutant macrophages within carrier mice than in non-mutant macrophages of non-carrier chimeras, and showed relatively small differences compared to Dnmt3a-deficient macrophages. Conclusions Our experimental findings in humans and mice support a pathomechanistic model in which few DNMT3A-CHIP mutation-carrying hematopoietic stem cells stimulate non-mutant cells within the bone marrow, thereby augmenting inflammation downstream in all circulating monocytes and their progeny in atherosclerotic lesions.