Bone Marrow Cells Research Articles

A Comprehensive Protocol for the Collection, Differentiation, Cryopreservation, and Resuscitation of Primary Murine Bone Marrow Derived Macrophages (BMDM)

ABSTRACT Background The field of immunology has undoubtedly benefited from the in vitro use of cell lines for immunological studies; however, due to the “immortal” nature of many cell lines, they are not always the best model. Thus, direct collection and culture of primary cells from model organisms is a solution that many researchers utilize. To the best of our knowledge, there is not a singular protocol which encompasses the entire process of bone marrow cell collection through cryopreservation and resuscitation of cells from a murine model. Methods Bone marrow cells were collected from mice with a C57BL6 genetic background. Cells were differentiated using L929 conditioned media. Cells were assessed using a combination of microscopy, differential staining, immunocytochemistry, and trypan blue. Results: Primary murine BMDMs that underwent cryopreservation followed by resuscitation retained a high degree of viability. Furthermore, these BMDMs retained on overall ability to clear S. aureus. Results Primary murine BMDMs that underwent cryopreservation followed by resuscitation retained a high degree of viability. Furthermore, these BMDMs retained on overall ability to clear S. aureus. Conclusion Crypopreserved and resuscitated primary murine BMDMs were viable and retained their pverall S. aureus clearance ability.

Tumor-derived GLI1 promotes remodeling of the immune tumor microenvironment in melanoma

BackgroundMelanoma progression is based on a close interaction between cancer cells and immune cells in the tumor microenvironment (TME). Thus, a better understanding of the mechanisms controlling TME dynamics and composition will help improve the management of this dismal disease. Work from our and other groups has reported the requirement of an active Hedgehog-GLI (HH-GLI) signaling for melanoma growth and stemness. However, the role of the downstream GLI1 transcription factor in melanoma TME remains largely unexplored.MethodsThe immune-modulatory activity of GLI1 was evaluated in a syngeneic B16F10 melanoma mouse model assessing immune populations by flow cytometry. Murine polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) were differentiated from bone marrow cells and their immunosuppressive ability was assessed by inhibition of T cells. Conditioned media (CM) from GLI1-overexpressing mouse melanoma cells was used to culture PMN-MDSCs, and the effects of CM were evaluated by Transwell invasion assay and T cell inhibition. Cytokine array analysis, qPCR and chromatin immunoprecipitation were performed to explore the regulation of CX3CL1 expression by GLI1. Human monocyte-derived dendritic cells (moDCs) were cultured in CM from GLI1-silenced patient-derived melanoma cells to assess their activation and recruitment. Blocking antibodies anti-CX3CL1, anti-CCL7 and anti-CXCL8 were used for in vitro functional assays.ResultsMelanoma cell-intrinsic activation of GLI1 promotes changes in the infiltration of immune cells, leading to accumulation of immunosuppressive PMN-MDSCs and regulatory T cells, and to decreased infiltration of dendric cells (DCs), CD8 + and CD4 + T cells in the TME. In addition, we show that ectopic expression of GLI1 in melanoma cells enables PMN-MDSC expansion and recruitment, and increases their ability to inhibit T cells. The chemokine CX3CL1, a direct transcriptional target of GLI1, contributes to PMN-MDSC expansion and recruitment. Finally, silencing of GLI1 in patient-derived melanoma cells promotes the activation of human monocyte-derived dendritic cells (moDCs), increasing cytoskeleton remodeling and invasion ability. This phenotype is partially prevented by blocking the chemokine CCL7, but not CXCL8.ConclusionOur findings highlight the relevance of tumor-derived GLI1 in promoting an immune-suppressive TME, which allows melanoma cells to evade the immune system, and pave the way for the design of new combination treatments targeting GLI1.

Abstract We073: CLEC4E Signaling in Myocardial Ischemia-Reperfusion Injury

Background: The innate immune receptor CLEC4E (C-type lectin domain family 4 member E) in cardiac resident cells and circulating leukocytes contributes to ischemic myocardial damage and adverse LV remodeling following ischemia reperfusion injury (I/R). Mice with loss of CLEC4E gene function show reduced cardiac injury and better LV remodeling but the mechanisms of this cardioprotection remain unknown. Aims: We investigated whether Clec4e gene function in cardiac resident cells vs infiltrating immune cells mediates cardioprotection during myocardial ischemia-reperfusion injury. Methods: We generated chimeric mice by IV infusion of 5 x 10 6 bone marrow cells originating from C57Bl6/J CLEC4E -/- and C57Bl6/J wild-type (WT) donor mice in 8w-old lethally irradiated (9.5 Gy) WT (n=36) and CLEC4E -/- (n=50) receptor mice, respectively. After 4 w, mice were subjected to ischemia reperfusion injury (I/R) by 60 min LAD occlusion. Troponin levels (TnI) were measured at 90 min post I/R and 7T-magnetic resonance imaging with late gadolinium enhancement (LGE) for infarct size evaluation was performed at 24 h and 28 d post I/R. Mice were euthanized at 28 days for immunohistochemistry and analysis of BM chimerism. Results: Immunohistochemistry for CLEC4E expression on bone marrow smears confirmed successful BM chimerism. At 24h post I/R, TnI levels and LGE-assessed infarct size on MRI were not significantly different between groups (42.1 vs 48.4 ng/ml, and 25.2% vs 31.5% of LVmass in CLEC4E -/- & WT BM versus in WT & CLEC4E -/- BM, respectively) suggesting similar myocardial damage . After 28 d, LV end-diastolic volumes (43µL vs 55µL), and end-systolic volumes (13µL vs 25µL) were significantly smaller in CLEC4E -/- & WT BM than in WT & CLEC4E -/- BM mice (P<0.05 for both), resulting in better preserved global LV function (LVEF 62% vs 47%, P<0.05) . Infarct size, semiquantitatively assessed using planimetry on Sirius red stains, was smaller in CLEC4E -/- & WT BM than in WT & CLEC4E -/- BM (6.9% vs 19.8%, P<0.01). Conclusion: CLEC4E gene deletion in cardiac resident cells, but not in infiltrating hematopoietic immune cells is associated with better functional and structural remodelling after myocardial I/R. Inhibition of CLEC4E signaling via blocking antibodies or small molecule inhibitors in cardiac myocytes might be a promising cardioprotective strategy.

Correlation between Morphological Typing and Monoclonality of Bone Marrow Plasma Cells and Its Diagnostic Value for High-Risk Smoldering Multiple Myeloma

To investigate the correlation between morphological typing and monoclonality of bone marrow plasma cells, and explore the diagnostic value of plasma cell morphological typing for high-risk smoldering multiple myeloma(HR-SMM). The correlation between the morphological characteristics and the monoclonality of bone marrow plasma cells was analyzed in 84 patients with HR-SMM who treated in our hospital. The consistency of morphologically abnormal bone marrow plasma cells with serum free light chain (sFLC) ratio, next-generation sequencing (NGS) detection results, and its correlation with monoclonal plasma cells detected by flow cytometry (FCM) were further verified. The immunoglobulin types and levels of non-involved immunoglobulins in serum of the patients were detected, and the distribution of plasma cell clusters in patients with different disease was observed. The mean percentage of mature plasma cells were decreased successively in the order of reactive plasmacytosis (RP) group, monoclonal gammopathy of undetermined significance (MGUS) group, smoldering multiple myeloma (SMM) group, HR-SMM group and multiple myeloma (MM) group; while the mean percentage of immature, primitive, reticular and flaming plasma cells were increased successively in the order of RP group, MGUS group, SMM group, and HR-SMM group, and the difference between any two groups was statistically significant (P < 0.05).The average proportion of abnormal plasma cells in the bone marrow of HR-SMM patients was 96.2% of the total plasma cells. The proportion of abnormal plasma cells were in good agreement with the sFLC ratio and the results of NGS detection in HR-SMM patients (kappa=0.879 and kappa=0.891, both >0.75)，and showed good correlation with the monoclonal plasma cells with immunophenotype of CD45-/CD38+/CD138+/CD56+/CD19-( γ=0.825). The levels of non-involved immunoglobulin in IgG, IgA and IgM type HR-SMM patients were all decreased by more than 25% compared with the normal reference range, and the differences were statistically significant (P < 0.05). There was no significant difference in the distribution ratio of plasma cell clusters among different disease groups (P >0.05). In HR-SMM patients, the immature, primitive, reticular and flaming plasma cells in bone marrow are considered as abnormal plasma cells, and they are correlated with monoclonal plasma cells. The proportion of abnormal plasma cells in total plasma cells of bone marrow and the reduction extent of non-involved immunoglobulin level in patients have certain reference value for the diagnosis of HR-SMM.

Quantitative Detection and Integrality Analysis of Plasma Circulating Cell-Free DNA in Multiple Myeloma

To investigate the role of plasma circulating cell-free DNA (cf-DNA) in the screening and diagnosis of patients with newly diagnosed multiple myeloma (MM) and explore the changes of cf-DNA in terms of content and integrality in the assessment of disease in patients treated with chemotherapy. Peripheral blood specimens were collected from 35 newly diagnosed MM patients and 18 healthy volunteers, and 13 of the 35 patients who had finished 3 courses of standard induction chemotherapy were selected as follow-up group. The ALU247 and ALU115 fragments were used as the target genes, and the cf-DNA content in the plasma of patients and healthy controls was measured by quantitative polymerase chain reaction (qPCR). The integrality of cf-DNA was calculated by the ratio of ALU247 to ALU115. Both the concentration of ALU247 and ALU115 fragments and the integrality of cf-DNA in patients were significantly higher than those in healthy controls (all P < 0.05). Patients who had underwent 3 courses of induction chemotherapy had significantly decreased concentration of ALU247 and ALU115 fragments and integrality of cf-DNA after treatment (all P < 0.05), and strong positive correlations were manifested between cf-DNA integrality and serum M protein content, as well as proportion of abnormal plasma cells in bone marrow before and after treatment (r =0.703, 0.705). Cf-DNA has certain positive significance for the screening and diagnosis of MM. Furthermore, cf-DNA may be a synergism or alternative to serum M-protein content and proportion of abnormal plasma cells in bone marrow in assessing the condition, curative effect and prognosis of patients.

Identification of critical genes associated with oxidative stress pathways in benzene-induced hematotoxicity

Background and aimsBone marrow failure (BMF) is chronic benzene-induced hematotoxicity, which is associated with differential gene expression abnormality. Benzene-induced BMF is characterized by irreversible bone marrow depression. Despite extensive studies have been conducted, there is a lack of reliable, useful and simple diagnostic method for BMF. Previous studies have shown that the aberrant gene expression changes and reactive oxygen species production in bone marrow cells related to the development of BMF. Early detection of differentially expressed genes (DEGs) as potential biomarkers is important for diagnosis and treatment. However, the validation of effective biomarker through DEGs analysis in benzene-induced BMF still deserve to be clarified. This study aimed to identify target genes as potential biomarkers with benzene-induced BMF based on DEGs analysis. MethodsFirst, we developed a benzene-induced BMF mouse model and obtained the DEGs in bone marrow cells of benzene-exposed CD1 mice. Next, after obtaining the DEGs via RNA-Sequencing (RNA-seq) analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were also used, key genes associated with benzene-induced BMF were identified. Additionally, the key markers for benzene poisoning was evaluated using qRT-PCR technique. ResultsWe identified DEGs for further KEGG functional analysis. Ten statistically significantly (up or down) regulated genes, namely Mapk11, Foxo1, Lefty1, Ren1, Bank1, Fgf3, Cdc42ep2, Rasgrf1, P2rx7, and Shank3 were found mainly associated with mitogen-activated protein kinases (MAPK) oxidative stress pathway. Further analysis using qRT-PCR identified that eight statistically significant DEGs associated with signaling pathways such as MAPK. We found that the level of mRNA expression of Mapk11, Foxo1, Bank1, Lefty1, Ren1, P2rx7, and Fgf3 genes were increased and Cdc42ep2 gene was decreased in BMF mice compared to control mice. Additionally, we validated the eight candidate genes for potential biomarkers in peripheral blood mononuclear cells of benzene poisoning patients by qRT-PCR. ConclusionOur results indicated that Mapk11 and Fgf3 were predominantly candidate genes linked to novel biomarkers for benzene hematotoxicity in human beings. Our study will provide new candidate genes as useful biomarkers involved in benzene-induced hematotoxicity.

Characterization of Turbo, a TLR Ligand-based Adjuvant for Glycoconjugate Vaccines.

Many bacterial polysaccharide vaccines, including the typhoid Vi polysaccharide (ViPS) and tetravalent meningococcal polysaccharide conjugate (MCV4) vaccines, do not incorporate adjuvants and are not highly immunogenic, particularly in infants. I found that endotoxin, a TLR4 ligand in ViPS, contributes to the immunogenicity of typhoid vaccines. Because endotoxin is pyrogenic, and its levels are highly variable in vaccines, I developed monophosphoryl lipid A, a nontoxic TLR4 ligand-based adjuvant named Turbo. Admixing Turbo with ViPS and MCV4 vaccines improved their immunogenicity across all ages and eliminated booster requirement. To understand the characteristics of this adjuvanticity, I compared Turbo with alum. Unlike alum, which polarizes the response toward the IgG1 isotype, Turbo promoted Ab class switching to all IgG isotypes with affinity maturation; the magnitude of this IgG response is durable and accompanied by the presence of long-lived plasma cells in the mouse bone marrow. In striking contrast with the pathways employed by alum, Turbo adjuvanticity is independent of NLPR3, pyroptotic cell death effector Gasdermin D, and canonical and noncanonical inflammasome activation mediated by Caspase-1 and Caspase-11, respectively. Turbo adjuvanticity is primarily dependent on the MyD88 axis and is lost in mice deficient in costimulatory molecules CD86 and CD40, indicating that Turbo adjuvanticity includes activation of these pathways. Because Turbo formulations containing either monophosphoryl lipid A or TLR2 ligands, Pam2CysSerLys4, and Pam3CysSerLys4 help generate Ab response of all IgG isotypes, as an adjuvant Turbo can improve the immunogenicity of glycoconjugate vaccines against a wide range of bacterial pathogens whose elimination requires appropriate IgG isotypes.

Effects of PM2.5 exposure on hematopoiesis and coupled immune disorder in adult male mice

Fine particulate matter, namely PM2.5, increases the risk of morbidity and mortality in various systems, including the hematopoietic and immune systems. However, there is limited experimental evidence supporting the association between PM2.5 exposure and hematopoietic outcomes as most studies focus on epidemiological data. In this study, adult male mice were exposed to PM2.5 for a long time to investigate hematopoietic toxicity. There were significant increases in red blood cells (RBC), hemoglobin (HGB), and white blood cells (WBC) in peripheral blood after 5-month real-environmental PM2.5 exposure, indicating elevated circulatory inflammation and potential hematopoietic abnormality. Moreover, we observed abnormal proliferation and differentiation of hematopoietic stem and progenitor cells (HSPCs), along with altered mRNA levels of hematopoietic genes and increased proinflammatory factors in the bone marrow (BM). Transcriptomic analysis of BM cells suggested that PM2.5 exposure activated immune responses associated with lymphocytes. Then, PM2.5 exposure via intratracheal instillation was performed for 8 weeks to verify the toxic effect. The results of the complete blood count were similar to those of real-environmental exposure, with drastic changes in the number and function of HSPCs, as well as mRNA levels of the hematopoietic genes and increased inflammatory factors in the BM being detected. Furthermore, lymphocyte subsets changed significantly in the BM and spleen, confirming immune disorder following PM2.5 exposure. In conclusion, PM2.5 interfered with the process of BM hematopoiesis by triggering inflammation and leading to immune disorder. Our study provided experimental evidence for the hematopoietic toxicity of PM2.5 and highlighted the significance of reducing air pollution.

Prostacyclin Synthase Deficiency Leads to Exacerbation or Occurrence of Endothelium-Dependent Contraction and Causes Cardiovascular Disorders Mainly via the Non-TxA2 Prostanoids/TP Axis.

Prostaglandin I2 synthesized by endothelial COX (cyclooxygenase) evokes potent vasodilation in some blood vessels but is paradoxically responsible for endothelium-dependent constriction (EDC) in others. Prostaglandin I2 production and EDC may be enhanced in diseases such as hypertension. However, how PGIS (prostaglandin I2 synthase) deficiency affects EDC and how this is implicated in the consequent cardiovascular pathologies remain largely unknown. Experiments were performed with wild-type, Pgis knockout (Pgis-/-) and Pgis/thromboxane-prostanoid receptor gene (Tp) double knockout (Pgis-/-Tp-/-) mice and Pgis-/- mice transplanted with unfractionated wild-type or Cox-1-/- bone marrow cells, as well as human umbilical arteries. COX-derived prostanoids were measured by high-performance liquid chromatography-mass spectrometry. Vasomotor responses of distinct types of arteries were assessed by isometric force measurement. Parameters of hypertension, vascular remodeling, and cardiac hypertrophy in mice at different ages were monitored. PGF2α, PGE2, and a trace amount of PGD2, but not thromboxane A2 (TxA2), were produced in response to acetylcholine in Pgis-/- or PGIS-inhibited arteries. PGIS deficiency resulted in exacerbation or occurrence of EDC ex vivo and in vivo. Endothelium-dependent hyperpolarization was unchanged, but phosphorylation levels of eNOS (endothelial nitric oxide synthase) at Ser1177 and Thr495 were altered and NO production and the NO-dependent relaxation evoked by acetylcholine were remarkably reduced in Pgis-/- aortas. Pgis-/- mice developed high blood pressure and vascular remodeling at 16 to 17 weeks and subsequently cardiac hypertrophy at 24 to 26 weeks. Meanwhile, blood pressure and cardiac parameters remained normal at 8 to 10 weeks. Additional ablation of TP (TxA2 receptor) not only restrained EDC and the downregulation of NO signaling in Pgis-/- mice but also ameliorated the cardiovascular abnormalities. Stimulation of Pgis-/- vessels with acetylcholine in the presence of platelets led to increased TxA2 generation. COX-1 disruption in bone marrow-derived cells failed to affect the development of high blood pressure and vascular remodeling in Pgis-/- mice though it largely suppressed the increase of plasma TxB2 (TxA2 metabolite) level. Our study demonstrates that the non-TxA2 prostanoids/TP axis plays an essential role in mediating the augmentation of EDC and cardiovascular disorders when PGIS is deficient, suggesting TP as a promising therapeutic target in diseases associated with PGIS insufficiency.

Prognostic Significance of Disseminated Tumor Cells in Bone Marrow for Endometrial Carcinoma Patients.

Background: Until now, limited clinical significance had been reported for disseminated tumor cells (DTCs) in gynecologic malignancies. DTCs were previously reported not to be associated with established risk factors, L1CAM immunoreactivity, and outcome in endometrial carcinoma (EC). This study's primary objective was to investigate potential correlations of DTCs in the bone marrow (BM) of EC patients with disease-related survival, and a secondary objective was to evaluate associations between molecular classification of EC and DTCs. Methods: Patients treated for primary EC at Tuebingen University women's hospital between 2003 and 2016 were identified. A total of 402 patients with a complete set of BM cytology, molecular, and clinical data were evaluable. Results: DTC occurrence was distributed equally among all four molecular groups (p = 0.651). DTC positivity was associated with a less favorable disease-free survival (HR: 1.86, 95% CI: 1.03-3.36, p = 0.036) and progression-free survival (HR: 1.86, 95% CI: 1.01-3.44, p = 0.045). Presence of DTCs was associated with a higher frequency of distant disease recurrence (p = 0.017). Conclusions: In line with our previous findings, tumor cell dissemination is not associated with molecular features in our large cohort of primary EC patients. Since DTCs seem to be associated with survival and location of disease recurrence, further studies are needed to decisively define their role in EC survival.

Potential Targeting Mechanisms for Bone-Directed Therapies.

Bone development is characterized by complex regulation mechanisms, including signal transduction and transcription factor-related pathways, glycobiological processes, cellular interactions, transportation mechanisms, and, importantly, chemical formation resulting from hydroxyapatite. Any abnormal regulation in the bone development processes causes skeletal system-related problems. To some extent, the avascularity of cartilage and bone makes drug delivery more challenging than that of soft tissues. Recent studies have implemented many novel bone-targeting approaches to overcome drawbacks. However, none of these strategies fully corrects skeletal dysfunction, particularly in growth plate-related ones. Although direct recombinant enzymes (e.g., Vimizim for Morquio, Cerezyme for Gaucher, Elaprase for Hunter, Mepsevii for Sly diseases) or hormone infusions (estrogen for osteoporosis and osteoarthritis), traditional gene delivery (e.g., direct infusion of viral or non-viral vectors with no modifications on capsid, envelope, or nanoparticles), and cell therapy strategies (healthy bone marrow or hematopoietic stem cell transplantation) partially improve bone lesions, novel delivery methods must be addressed regarding target specificity, less immunogenicity, and duration in circulation. In addition to improvements in bone delivery, potential regulation of bone development mechanisms involving receptor-regulated pathways has also been utilized. Targeted drug delivery using organic and inorganic compounds is a promising approach in mostly preclinical settings and future clinical translation. This review comprehensively summarizes the current bone-targeting strategies based on bone structure and remodeling concepts while emphasizing potential approaches for future bone-targeting systems.

The Phoenix of stem cells: pluripotent cells in adult tissues and peripheral blood.

Pluripotent stem cells are defined as cells that can generate cells of lineages from all three germ layers, ectoderm, mesoderm, and endoderm. On the contrary, unipotent and multipotent stem cells develop into one or more cell types respectively, but their differentiation is limited to the cells present in the tissue of origin or, at most, from the same germ layer. Multipotent and unipotent stem cells have been isolated from a variety of adult tissues, Instead, the presence in adult tissues of pluripotent stem cells is a very debated issue. In the early embryos, all cells are pluripotent. In mammalians, after birth, pluripotent cells are maintained in the bone-marrow and possibly in gonads. In fact, pluripotent cells were isolated from marrow aspirates and cord blood and from cultured bone-marrow stromal cells (MSCs). Only in few cases, pluripotent cells were isolated from other tissues. In addition to have the potential to differentiate toward lineages derived from all three germ layers, the isolated pluripotent cells shared other properties, including the expression of cell surface stage specific embryonic antigen (SSEA) and of transcription factors active in the early embryos, but they were variously described and named. However, it is likely that they are part of the same cell population and that observed diversities were the results of different isolation and expansion strategies. Adult pluripotent stem cells are quiescent and self-renew at very low rate. They are maintained in that state under the influence of the "niche" inside which they are located. Any tissue damage causes the release in the blood of inflammatory cytokines and molecules that activate the stem cells and their mobilization and homing in the injured tissue. The inflammatory response could also determine the dedifferentiation of mature cells and their reversion to a progenitor stage and at the same time stimulate the progenitors to proliferate and differentiate to replace the damaged cells. In this review we rate articles reporting isolation and characterization of tissue resident pluripotent cells. In the attempt to reconcile observations made by different authors, we propose a unifying picture that could represent a starting point for future experiments.

Oral health management in children with severe congenital neutropenia with periodontitis: Case report.

Severe congenital neutropenia (SCN) is a rare and heterogeneous genetic disease. By describing the diagnosis and treatment of a child with SCN and periodontitis, this case provides a reference for the oral health management of a child with SCN and periodontitis. We describe a boy with clinical manifestations of oral bleeding, neutropenia, recurrent fever, and other recurrent infections. The absolute neutrophil count (ANC) was <0.50 × 109/L most of the time. Morphological examination of bone marrow cells showed active granulocyte hyperplasia and dysmaturation. According to the clinical manifestations, hematological examination and gene detection results, the child was diagnosed as SCN with chronic periodontitis. Periodontal treatment was performed after informed consent was obtained from the child guardian. These included supragingival and subgingival cleaning, hydrogen peroxide and saline irrigation, placement of iodoglycerin in the gingival sulcus, and oral hygiene instruction. Hematopoietic stem cell transplantation (HSCT) was performed later. One month after initial periodontal treatment, oral hygiene was well maintained and gingival swelling had subsided. Probing depth (PD) index on periodontal probing and bleeding was significantly reduced. However, there was no significant change in blood routine and other indicators before and after periodontal treatment. Once SCN is diagnosed, individualized treatment plans can be developed according to the characteristics of the disease and its impact on oral health, which can effectively control the interaction between SCN and periodontal disease and reduce the occurrence of serious infection.

Blinatumomab for MRD-Negative Acute Lymphoblastic Leukemia in Adults

BackgroundMany older adults with B-cell precursor acute lymphoblastic leukemia (BCP-ALL) have a relapse despite having a measurable residual disease (MRD)–negative complete remission with combination chemotherapy. The addition of blinatumomab, a bispecific T-cell engager molecule that is approved for the treatment of relapsed, refractory, and MRD-positive BCP-ALL, may have efficacy in patients with MRD-negative remission.MethodsIn a phase 3 trial, we randomly assigned patients 30 to 70 years of age with BCR::ABL1-negative BCP-ALL (with :: indicating fusion) who had MRD-negative remission (defined as <0.01% leukemic cells in bone marrow as assessed on flow cytometry) after induction and intensification chemotherapy to receive four cycles of blinatumomab in addition to four cycles of consolidation chemotherapy or to receive four cycles of consolidation chemotherapy alone. The primary end point was overall survival, and relapse-free survival was a secondary end point.ResultsThe data and safety monitoring committee reviewed the results from the third efficacy interim analysis and recommended that they be reported. Complete remission with or without full count recovery was observed in 395 of 488 enrolled patients (81%). Of the 224 patients with MRD-negative status, 112 were assigned to each group. The characteristics of the patients were balanced between the groups. At a median follow-up of 43 months, an advantage was observed in the blinatumomab group as compared with the chemotherapy-only group with regard to overall survival (at 3 years: 85% vs. 68%; hazard ratio for death, 0.41; 95% confidence interval [CI], 0.23 to 0.73; P=0.002), and the 3-year relapse-free survival was 80% with blinatumomab and 64% with chemotherapy alone (hazard ratio for relapse or death, 0.53; 95% CI, 0.32 to 0.87). A higher incidence of neuropsychiatric events was reported in the blinatumomab group than in the chemotherapy-only group.ConclusionsThe addition of blinatumomab to consolidation chemotherapy in adult patients in MRD-negative remission from BCP-ALL significantly improved overall survival. (Funded by the National Institutes of Health and others; E1910 ClinicalTrials.gov number, NCT02003222.)

Polysaccharide from Helianthus tuberosus L. as a potential radioprotector

IntroductionRadioprotectors help to protect the body or at least minimize the negative consequences of radiation exposure. The present study aimed to assess the radioprotective potential of Helianthus tuberosus L. polysaccharide (HTLP) in vitality and micronuclei tests. To assess the cytotoxic effects of HTLP, both vitality and MTT reductase assays were conducted. Materials and methodsRAW 264.7 cells viability was assessed 24 h after adding 200 μg/ml HTLP solution by staining cell cultures with propidium iodide and bis-benzimide to detect the nuclei of dead cells and the total number of cells in culture. To assess cell viability via cellular metabolic activity MTT test was used.In this work outbred 24–30 g 5-months old SHK mice have been used. Irradiation was provided with proton beams with an energy of 660 MeV at a dose rate of 80 Gy with doses 1.5 Gy for micronuclei test and 8.5 Gy for survival test. Whole body X-ray irradiation was conducted using the RUT-15 therapeutic X-ray unit with doses of 1.5 Gy for MN test and 6.5 Gy for survival. The HTLP sterile solution in dose 100 μg/animal was injected into the tail vein 15 min before X-ray or proton irradiation. Results and conclusions: Vitality test showed no significant differences between the control group and cells treated with 200 μl of 200 μg/ml HTLP solution, though a greater variability was noted. In contrast, the MTT assay indicated enhanced cell viability in the HTLP-treated cells. HTLP does not exert any toxic effects in cell culture. Moreover, results of MTT reductase assay shows, that HTLP may enhance the cells’ metabolic activity.Animals pre-treated with HTLP displayed a significant reduction in micronuclei formation, showing five times fewer micronuclei in bone marrow cells compared to the non-treated group. This comparison highlights HTLP's potential protective effect against radiation-induced chromosomal damage. HTLP treatment demonstrates a significant reduction in hazard compared to the control, indicating its protective effects against irradiation. Thus, it can be concluded that the use of HTLP increases the likelihood of animal survival under the ionizing effects of X-rays and protons. The survival analysis reveals that the HTLP-treated groups exhibit a higher survival rate compared to both the control and Cysteamine-treated groups, suggesting a significant protective effect of HTLP against irradiation, regardless of the type of irradiation (proton or X-ray) with p < 0.0001.

Trafficking circuit of CD8+ T cells between the intestine and bone marrow governs antitumour immunity.

Immunotherapy elicits a systemic antitumour immune response in peripheral circulating T cells. However, the T cell trafficking circuit between organs and their contributions to antitumour immunity remain largely unknown. Here we show in multiple mouse leukaemia models that high infiltration of leukaemic cells in bone marrow (BM) stimulates the transition of CD8+CD44+CD62L+ central memory T cells into CD8+CD44-CD62L- T cells, designated as inter-organ migratory T cells (TIM cells). TIM cells move from the BM to the intestine by upregulating integrin β7 and downregulating C-X-C motif chemokine receptor 3 during leukaemogenesis. Upon immunogenic chemotherapy, these BM-derived TIM cells return from the intestine to the BM through integrin α4-vascular cell adhesion molecule 1 interaction. Blocking C-X-C motif chemokine receptor 3 function boosts the immune response against leukaemia by enhancing T cell trafficking. This phenomenon can also be observed in patients with leukaemia. In summary, we identify an unrecognized intestine-BM trafficking circuit of T cells that contributes to the antitumour effects of immunogenic chemotherapy.

Donor Sex and Passage Conditions Influence MSC Osteogenic Response in Mineralized Collagen Scaffolds.

Contemporary tissue engineering efforts often seek to use mesenchymal stem cells (MSCs) due to their multi-potent potential and ability to generate a pro-regenerative secretome. While many have reported the influence of matrix environment on MSC osteogenic response, few have investigated the effects of donor and sex. Here, a well-defined mineralized collagen scaffold is used to study the influence of passage number and donor-reported sex on MSC proliferation and osteogenic potential. A library of bone marrow and adipose tissue-derived stem cells from eight donors to examine donor viability in osteogenic capacity in mineralized collagen scaffolds is obtained. MSCs displayed reduced proliferative capacity as a function of passage duration. Further, MSCs showed significant sex-associated variability in osteogenic capacity. Notably, MSCs from male donors displayed significantly higher cell proliferation while MSCs from female donors displayed significantly higher osteogenic response via increased alkaline phosphate activity, osteoprotegerin release, and mineral formation in vitro. The study highlights the essentiality of including donor-reported sex as an experimental variable and reporting culture expansion in future studies of biomaterial regenerative potential.

Computing cell state discriminates the aberrant hematopoiesis and activated microenvironment in Myelodysplastic syndrome (MDS) through a single cell genomic study

BackgroundMyelodysplastic syndrome (MDS) is a complicated hematopoietic malignancy characterized by bone marrow (BM) dysplasia with symptoms like anemia, neutropenia, or thrombocytopenia. MDS exhibits considerable heterogeneity in prognosis, with approximately 30% of patients progressing to acute myeloid leukemia (AML). Single cell RNA-sequencing (scRNA-seq) is a new and powerful technique to profile disease landscapes. However, the current available scRNA-seq datasets for MDS are only focused on CD34+ hematopoietic progenitor cells. We argue that using entire BM cell for MDS studies probably will be more informative for understanding the pathophysiology of MDS.MethodsFive MDS patients and four healthy donors were enrolled in the study. Unsorted cells from BM aspiration were collected for scRNA-seq analysis to profile overall alteration in hematopoiesis.ResultsStandard scRNA-seq analysis of unsorted BM cells successfully profiles deficient hematopoiesis in all five MDS patients, with three classified as high-risk and two as low-risk. While no significant increase in mutation burden was observed, high-risk MDS patients exhibited T-cell activation and abnormal myelogenesis at the stages between hematopoietic stem and progenitor cells (HSPC) and granulocyte–macrophage progenitors (GMP). Transcriptional factor analysis on the aberrant myelogenesis suggests that the epigenetic regulator chromatin structural protein-encoding gene HMGA1 is highly activated in the high-risk MDS group and moderately activated in the low-risk MDS group. Perturbation of HMGA1 by CellOracle simulated deficient hematopoiesis in mouse Lineage-negative (Lin-) BM cells. Projecting MDS and AML cells on a BM cell reference by our newly developed MarcoPolo pipeline intuitively visualizes a connection for myeloid leukemia development and abnormalities of hematopoietic hierarchy, indicating that it is technically feasible to integrate all diseased bone marrow cells on a common reference map even when the size of the cohort reaches to 1,000 patients or more.ConclusionThrough scRNA-seq analysis on unsorted cells from BM aspiration samples of MDS patients, this study systematically profiled the development abnormalities in hematopoiesis, heterogeneity of risk, and T-cell microenvironment at the single cell level.

Synergistic Enhancement of Chemotherapy-Induced Cell Death and Antitumor Efficacy against Tumoral T-Cell Lymphoblasts by IMMUNEPOTENT CRP.

T-cell malignancies, including T-cell acute lymphoblastic leukemia (T-ALL) and T-cell lymphoblastic lymphoma (T-LBL), present significant challenges to treatment due to their aggressive nature and chemoresistance. Chemotherapies remain a mainstay for their management, but the aggressiveness of these cancers and their associated toxicities pose limitations. Immunepotent CRP (ICRP), a bovine dialyzable leukocyte extract, has shown promise in inducing cytotoxicity against various cancer types, including hematological cancers. In this study, we investigated the combined effect of ICRP with a panel of chemotherapies on cell line models of T-ALL and T-LBL (CEM and L5178Y-R cells, respectively) and its impact on immune system cells (peripheral blood mononuclear cells, splenic and bone marrow cells). Our findings demonstrate that combining ICRP with chemotherapies enhances cytotoxicity against tumoral T-cell lymphoblasts. ICRP + Cyclophosphamide (CTX) cytotoxicity is induced through a caspase-, reactive oxygen species (ROS)-, and calcium-dependent mechanism involving the loss of mitochondrial membrane potential, an increase in ROS production, and caspase activation. Low doses of ICRP in combination with CTX spare non-tumoral immune cells, overcome the bone marrow-induced resistance to CTX cell death, and improves the CTX antitumor effect in vivo in syngeneic Balb/c mice challenged with L5178Y-R. This led to a reduction in tumor volume and a decrease in Ki-67 proliferation marker expression and the granulocyte/lymphocyte ratio. These results set the basis for further research into the clinical application of ICRP in combination with chemotherapeutic regimens for improving outcomes in T-cell malignancies.

Proteomic screening identifies PF4/Cxcl4 as a critical driver of myelofibrosis.

Despite increased understanding of the genomic landscape of Myeloproliferative Neoplasms (MPNs), the pathological mechanisms underlying abnormal megakaryocyte (Mk)-stromal crosstalk and fibrotic progression in MPNs remain unclear. We conducted mass spectrometry-based proteomics on mice with Romiplostim-dependent myelofibrosis to reveal alterations in signaling pathways and protein changes in Mks, platelets, and bone marrow (BM) cells. The chemokine Platelet Factor 4 (PF4)/Cxcl4 was up-regulated in all proteomes and increased in plasma and BM fluids of fibrotic mice. High TPO concentrations sustained in vitro PF4 synthesis and secretion in cultured Mks, while Ruxolitinib restrains the abnormal PF4 expression in vivo. We discovered that PF4 is rapidly internalized by stromal cells through surface glycosaminoglycans (GAGs) to promote myofibroblast differentiation. Cxcl4 gene silencing in Mks mitigated the profibrotic phenotype of stromal cells in TPO-saturated co-culture conditions. Consistently, extensive stromal PF4 uptake and altered GAGs deposition were detected in Romiplostim-treated, JAK2V617F mice and BM biopsies of MPN patients. BM PF4 levels and Mk/platelet CXCL4 expression were elevated in patients, exclusively in overt fibrosis. Finally, pharmacological inhibition of GAGs ameliorated in vivo fibrosis in Romiplostim-treated mice. Thus, our findings highlight the critical role of PF4 in the fibrosis progression of MPNs and substantiate the potential therapeutic strategy of neutralizing PF4-GAGs interaction.