Immature Hematopoietic Cells Research Articles

Transforming growth factor-β1 and vascular endothelial growth factor levels in senile acute myeloid leukemia and correlation with prognosis.

Acute myeloid leukemia (AML) is a disease in which immature hematopoietic cells accumulate in the bone marrow and continuously expand, inhibiting hematopoiesis. The treatment and prognosis of this disease have always been unsatisfactory. To investigate the correlation between vascular endothelial growth factor (VEGF) and transforming growth factor-β1 (TGFβ1) expression and prognosis in older adults with AML. This study enrolled 80 patients with AML (AML group), including 36 with complete response (AML-CR), 23 with partial response (AML-PR), and 21 with no response (AML-NR). The expression levels of VEGF and TGFβ1 were detected by reverse transcription polymerase chain reaction in bone marrow mononuclear cells isolated from 56 healthy controls. Kaplan-Meier analysis was performed to assess overall survival (OS) and progression- or disease-free survival (DFS). Prognostic risk factors were analyzed using a Cox proportional hazards model. The AML group showed a VEGF level of 2.68 ± 0.16. VEGF expression was lower in patients with AML-CR than those with AML-PR or AML-NR (P < 0.05). TGFβ1 expression in the AML group was 0.33 ± 0.05. Patients with AML-CR showed a higher TGFβ1 expression than those with AML-PR or AML-NR (P < 0.05). VEGF and TGFβ1 expression in patients with AML was significantly correlated with the counts of leukocytes, platelets, hemoglobin, and peripheral blood immature cells (P < 0.05); Kaplan-Meier survival analysis revealed that patients with high TGFβ1 expression had better OS and DFS than those with low TGFβ1 expression (P < 0.05), whereas patients with low VEGF levels showed better OS and DFS than those with high VEGF levels (P < 0.05). VEGF, TGFβ1, and platelet count were identified by the Cox proportional hazards model as independent risk factors for OS (P < 0.05), while VEGF, TGFβ1, and white blood cell count were independent risk factors for DFS (P < 0.05). Decreased VEGF expression and increased TGFβ1 expression in patients with AML provide valuable references for determining and individualizing clinical treatment strategies.

Clinical features of immature leukemias in children.

Early T-cell precursor acute lymphoblastic leukemia (ETP-ALL), mixed phenotypic acute leukemia (MPAL), and acute myeloid leukemia with minimal differentiation (AML-M0) all originate from immature hematopoietic progenitor cells and have a poor prognosis. We investigated the clinical characteristics of these immature leukemias in 17 children (ETP-ALL: 8, MPAL: 5, AML-M0: 4) at seven institutions. Clinical and laboratory findings were comparable across disease types. Eleven and six patients received ALL- and AML-oriented induction chemotherapy, with six and four achieving complete remission (CR), respectively. Five additional patients achieved CR after salvage with the other type of chemotherapy. Eight patients received hematopoietic cell transplantation (HCT) in first CR, and six survived without relapse. However, six of seven patients who did not receive HCT during first CR relapsed; all underwent HCT later, and only three survived. The 5-year event-free survival (EFS) and overall survival (OS) rate were 37% and 69%, respectively. Patients who achieved CR after induction chemotherapy and received HCT in first CR had favorable EFS and OS. Notably, all patients who received HCT in first CR survived 5years after diagnosis. Appropriate induction chemotherapy and HCT in first CR could improve the outcome of immature leukemias.

Evaluation of a machine-learning model based on laboratory parameters for the prediction of acute leukaemia subtypes: a multicentre model development and validation study in France

Acute leukaemias are life-threatening haematological cancers characterised by the infiltration of transformed immature haematopoietic cells in the blood and bone marrow. Prompt and accurate diagnosis of the three main acute leukaemia subtypes (ie acute lymphocytic leukaemia [ALL], acute myeloid leukaemia [AML], and acute promyelocytic leukaemia [APL]) is of utmost importance to guide initial treatment and prevent early mortality but requires cytological expertise that is not always available. We aimed to benchmark different machine-learning strategies using a custom variable selection algorithm to propose an extreme gradient boosting model to predict leukaemia subtypes on the basis of routine laboratory parameters. This multicentre model development and validation study was conducted with data from six independent French university hospital databases. Patients aged 18 years or older diagnosed with AML, APL, or ALL in any one of these six hospital databases between March 1, 2012, and Dec 31, 2021, were recruited. 22 routine parameters were collected at the time of initial disease evaluation; variables with more than 25% of missing values in two datasets were not used for model training, leading to the final inclusion of 19 parameters. The performances of the final model were evaluated on internal testing and external validation sets with area under the receiver operating characteristic curves (AUCs), and clinically relevant cutoffs were chosen to guide clinical decision making. The final tool, Artificial Intelligence Prediction of Acute Leukemia (AI-PAL), was developed from this model. 1410 patients diagnosed with AML, APL, or ALL were included. Data quality control showed few missing values for each cohort, with the exception of uric acid and lactate dehydrogenase for the cohort from Hôpital Cochin. 679 patients from Hôpital Lyon Sud and Centre Hospitalier Universitaire de Clermont-Ferrand were split into the training (n=477) and internal testing (n=202) sets. 731 patients from the four other cohorts were used for external validation. Overall AUCs across all validation cohorts were 0·97 (95% CI 0·95-0·99) for APL, 0·90 (0·83-0·97) for ALL, and 0·89 (0·82-0·95) for AML. Cutoffs were then established on the overall cohort of 1410 patients to guide clinical decisions. Confident cutoffs showed two (0·14%) wrong predictions for ALL, four (0·28%) wrong predictions for APL, and three (0·21%) wrong predictions for AML. Use of the overall cutoff greatly reduced the number of missing predictions; diagnosis was proposed for 1375 (97·5%) of 1410 patients for each category, with only a slight increase in wrong predictions. The final model evaluation across both the internal testing and external validation sets showed accuracy of 99·5% for ALL diagnosis, 98·8% for AML diagnosis, and 99·7% for APL diagnosis in the confident model and accuracy of 87·9% for ALL diagnosis, 86·3% for AML diagnosis, and 96·1% for APL diagnosis in the overall model. AI-PAL allowed for accurate diagnosis of the three main acute leukaemia subtypes. Based on ten simple laboratory parameters, its broad availability could help guide initial therapies in a context where cytological expertise is lacking, such as in low-income countries. None.

HSPA8 Chaperone Complex Drives Chaperone-Mediated Autophagy Regulation in Acute Promyelocytic Leukemia Cell Differentiation

Introduction: Acute myeloid leukemia (AML) is a cancer of the hematopoietic system characterized by hyperproliferation of undifferentiated cells of the myeloid lineage. While most of AML therapies are focused toward tumor debulking, all-trans retinoic acid (ATRA) induces neutrophil differentiation in the AML subtype acute promyelocytic leukemia (APL). Macroautophagy has been extensively investigated in the context of various cancers and is often dysregulated in AML where it can have context-dependent pro- or anti-leukemogenic effects. On the contrary, the implications of chaperone-mediated autophagy (CMA) on the pathophysiology of diseases are still being explored and its role in AML remains elusive. Methods: We took advantage of human AML primary samples and databases to analyze CMA gene expression and activity. Furthermore, we used ATRA-sensitive (NB4) and -resistant (NB4-R1) APL cells to further dissect a potential function for CMA in ATRA-mediated neutrophil differentiation. NB4-R1 cells are unique in that they do respond to retinoic acid transcriptionally but do not mature in response to retinoid signaling alone unless maturation is triggered by adding cyclic adenosine monophosphate. Results: Here, we report that CMA-related mRNA transcripts are significantly higher expressed in immature hematopoietic cells as compared to neutrophils, contrasting the macroautophagy gene expression patterns. Accordingly, lysosomal degradation of an mCherry-KFERQ CMA reporter decreases during ATRA-induced differentiation of APL cells. On the other hand, using NB4-R1 cells we found that macroautophagy flux primed ATRA-resistant NB4-R1 cells to differentiate upon ATRA treatment but reduced the association of lysosome-associated membrane protein type 2A (LAMP-2A) and heat shock protein family A (Hsp70) member 8 (HSPA8), necessary for complete neutrophil maturation. Accordingly, depletion of HSPA8 attenuated CMA activity and facilitated APL cell differentiation. In contrast, maintaining high CMA activity by ectopic expression of LAMP-2A impeded APL differentiation. Conclusion: Overall, our findings suggest that APL neutrophil differentiation requires CMA inactivation and that this pathway predominantly depends on HSPA8 and is possibly assisted by other co-chaperones.

Utilizing exosomes as sparking clinical biomarkers and therapeutic response in acute myeloid leukemia.

Acute myeloid leukemia (AML) is a malignant clonal tumor originating from immature myeloid hematopoietic cells in the bone marrow with rapid progression and poor prognosis. Therefore, an in-depth exploration of the pathogenesis of AML can provide new ideas for the treatment of AML. In recent years, it has been found that exosomes play an important role in the pathogenesis of AML. Exosomes are membrane-bound extracellular vesicles (EVs) that transfer signaling molecules and have attracted a large amount of attention, which are key mediators of intercellular communication. Extracellular vesicles not only affect AML cells and normal hematopoietic cells but also have an impact on the bone marrow microenvironment and immune escape, thereby promoting the progression of AML and leading to refractory relapse. It is worth noting that exosomes and the various molecules they contain are expected to become the new markers for disease monitoring and prognosis of AML, and may also function as drug carriers and vaccines to enhance the treatment of leukemia. In this review, we mainly summarize to reveal the role of exosomes in AML pathogenesis, which helps us elucidate the application of exosomes in AML diagnosis and treatment.

Expression, Function, and Significance of NonB Cell-Derived Immunoglobulin in Haematological System.

Acute myeloid leukaemia (AML) is a collection of genetically diverse diseases characterised by abnormal proliferation of immature haematopoietic cells and disruption of normal haematopoiesis. Myeloid cells and lymphocytes originate from different haematopoietic precursors within the bone marrow. It has been traditionally assumed that myeloid cells cannot produce immunoglobulin (Ig), a marker of B cells and plasma cells. However, in recent years, all five Ig classes have been detected in CD34+ haematopoietic stem cells, mature monocytes and neutrophils, differentiated macrophages and tumour-associated macrophages, acute myeloid leukaemia cell lines, as well as myeloblasts of AML. The rearranged V(D)J sequences exhibit unique restricted or biased V gene usage and evidence of somatic mutation. Furthermore, AML-derived Igs could promote cell proliferation, induce apoptosis, and enhance migration. Elevated levels of Ig expression predict inferior clinical outcomes. These findings indicate that AML-derived Ig plays a role in AML pathogenesis and progression, and could serve as a novel biomarker for risk stratification, disease monitoring, and targeted therapy. In this chapter, we provide a comprehensive review of recent literature on the expression, function, and significance of non B cell-derived Ig in the haematological system, with a focus on AML.

Estimation of plasma soluble interleukin-2 receptor alpha chain level in adults with acute myeloid leukemia

Abstract: BACKGROUND: Acute myeloid leukemia (AML) is a clonal malignant condition of immature hematopoietic cells, characterized by clonal proliferation of abnormal cells (blasts) in the marrow leading to impairment of the normal blood cell production giving rise to failure of the bone marrow. Soluble interleukin-2 (IL-2) receptor alpha chain is a protein that is involved in the assembly of the high-affinity IL-2 receptor, and it has a critical role in controlling immune system homeostasis. The overexpression of sIL-2RA was investigated in many hematopoietic malignancies, and it was correlated with poor outcome. OJECTIVES: The aim of this study was to assess the sIL-2RA level as a prognostic factor and assess its impact on survival and if it can be used as a targeted treatment for a better outcome. MATERIALS AND METHODS: Sixty newly diagnosed adults with AML before starting therapy were included in the study, and they were followed up for 6 months to document survival status. Thirty healthy adults were taken as a control group. Using an enzyme-linked immunosorbent assay, the plasma sIL-2RA level was measured. Statistical analysis was done using Microsoft Excel 2019 and version 26 SPSS statistical software. P &lt;0.05 was considered statistically significant. RESULTS: A considerable difference in the plasma sIL-2RA level between AML patients and controls also was more elevated in patients who died after 6-month follow-up. According to the blast percentage, total white blood cell count, and M0-M2 subgroups, the sIL-2RA level correlated positively. Irrelevant association was found regarding the patients’ age, the count of platelet, and the hemoglobin. CONCLUSIONS: Plasma sIL-2RA level is higher in AML patients than the control group at the time of diagnosis. Patients with a high level of plasma sIL-2RA have an inferior (overall survival) and poor outcome. SIL-2RA level is higher in M0-M2 subgroups than other subtypes. There is a significant association between sIL-2RA level and the absolute count of leukemic blasts.

Effects of Black Raspberry Supplementation on Methylation Pathways in Vav-creAsxl1fl/flTet2fl/fl Double Knockout Mice with Early-stage Myelodysplastic Syndrome.

Myelodysplastic syndromes (MDS) are a subset of myeloid malignancies defined by clonality of immature hematopoietic stem cells that leads to faulty blood cell development. These syndromes can lead to an increased risk of infection and may transform into acute myeloid leukemia, making it critical to determine effective treatments for the condition. While hypomethylating agents such as azacitidine and decitabine, as well as stem cell transplants, have been delineated as favored treatments for MDS, not all patients are physiologically receptive to these treatments. However, black raspberries (BRBs) have been shown to exert hypomethylating effects in various malignancies, with minimal adverse effects and thus a broader range of potential candidacies. This study aimed to investigate the potential of BRBs to exert such effects on MDS using Addition of Sex Combs Like/Tet Methylcytosine Dioxygenase 2 (Asxl1/Tet2) double knockout mice (Vav-cre Asxl1fl/fl Tet2fl/fl), which typically manifest symptoms around 25 weeks of age, mirroring genetic mutations found in humans with MDS. Following a 12-week dietary supplementation of Vav-cre Asxl1fl/fl Tet2fl/fl mice with 5% BRBs, we observed both hyper- and hypomethylation at multiple transcription start sites and intragenic locations linked to critical pathways, including hematopoiesis. This methylation profile may have implications for delaying the onset of MDS, prompting a need for in-depth investigation. Our results emphasize the importance of exploring whether an extended BRB intervention can effectively alter MDS risk and elucidate the relationship between BRB-induced methylation changes, thus further unlocking the potential benefits of BRBs for MDS patients.

TNFα-Driven Interplay between Hematopoietic Stem Cells and Antigen-Specific Cytotoxic T Cells

Clonal hematopoiesis (CH) is governed by aberrant hematopoietic stem cell (HSC) clones with leukemia-associated somatic mutations that arise spontaneously during organismal aging or after exposure to genotoxic insults. Cells with a somatic mutation can be eliminated through presentation of neoantigens on MHC class I (MHC-I) to antigen-specific cytotoxic T cells. Indeed, many of the CH-related mutations, such as JAK2V617F, are reported or predicted to elicit antigen-specific cytotoxic T cell response. On the other hand, accumulating evidence suggests that inflammatory environment facilitates clonal expansion of abnormal HSCs. However, much remains to be elucidated on whether HSCs expressing such non-self peptides can be eliminated by antigen-specific cytotoxic T cells and how inflammation affects the T cell-mediated HSC regulation. Our RNA-seq analyses revealed that genes involved in MHC-I-dependent antigen presentations, such as Nlrc5, H2-kb and B2m, are most abundantly expressed in HSCs (Lin -/c-Kit +/Sca-1 +/Flk2 -/CD48 -/CD150 +) among murine bone marrow immature hematopoietic cells. Using transgenic mice that ubiquitously express a model antigen ovalbumin (OVA) and a monoclonal antibody that specifically recognizes MHC-I-OVA complex, we found that while MHC-I protein is abundantly expressed on the surface of hematopoietic cells except granulocytes, HSCs and multipotent progenitor cells (MPPs) possess greater capacity to present intracellular antigens through MHC-I compared to myeloid progenitor and mature cells. Remarkably, in vitro co-culture experiments revealed that whereas OVA-expressing (OVA +) HSCs, MPPs and myeloid progenitors could directly activate OVA-specific OT-I CD8 + T cells through MHC-I-dependent antigen presentation, HSCs are the most susceptible population to the killing effect by cytotoxic T cells. Indeed, co-infused OT-I CD8 + T cells totally abolished the long-term repopulation capacity of OVA + HSCs in a serial transplantation setting, and adoptive transfer of OT-I CD8 + T cells to mixed chimeric mice that harbor both wild-type and OVA + hematopoietic cells completely and specifically eliminated hematopoiesis governed by OVA + HSCs. TNFα is not only a prototypical inflammatory cytokine but also one of the major cytokines that are secreted by CD8 + cytotoxic T cells. Notably, genes directly involved in T cell regulation including Cxcl9, Cd274, and Pdcd1lg2 were induced in HSCs, but not myeloid progenitor cells, upon exposure to TNFα (Yamashita and Passegué, Cell Stem Cell 2019). Indeed, upon co-culturing OT-I CD8 + T cells produced TNFα as well as IFNγ and Granzyme B and OVA + HSCs but not myeloid progenitor cells produced CXCL9 largely in a TNFα receptor-dependent manner. Interestingly, such HSC response to T cell-derived TNFα was critical for naïve CD8 + T cells to rapidly differentiate to effector cytotoxic T cells, as TNFα receptor-deficient ( Tnfr1 -/-Tnfr2 -/-) OVA + HSCs stimulated proliferation and cytokine production of OT-I CD8 + T cells but failed to stimulate Granzyme B production, whereas IFNγ receptor deficiency ( Ifngr -/-) did not have such effect. As a result, Tnfr1 -/-Tnfr2 -/- OVA + HSCs, but not Ifngr -/- OVA + HSCs, could escape from the killing by OT-I CD8 +T cells. In sharp contrast, prior exposure to environmental TNFα, but not to IL-1β or IL-6, before interaction with OT-I CD8 + T cells rendered OVA + HSCs resistant to the killing by OT-I CD8 + T cells largely through upregulation of PD-L1 and PD-L2. Of note, upon 24-month aging or acquisition of Jak2V617F mutation, OVA + HSCs still maintained robust MHC-I-dependent antigen presentation activity and high susceptibility to the killing by OT-I CD8 + T cells, though prior exposure to TNFα allowed for their immune escape. Consequently, adoptive transfer of OT-I CD8 + T cells completely and specifically eliminated Jak2V617F-mutant OVA + HSCs and reversed myeloproliferative neoplasms in the mixed chimeric mice. Taken together, our results reveal the robustness of HSC quality control via MHC-I-dependent antigen presentation to antigen-specific cytotoxic T cells. They also highlight a critical but complex role of TNFα in active interplay between HSCs and antigen-specific cytotoxic T cells as well as immune evasion by HSCs during inflammation. Specifically targeting abnormal HSCs by antigen-specific cytotoxic T cells may pave the way to eradicate mutant HSC clones and prevent CH-associated disease.

Screening for Intracellular Phosphorylation Cascades That Positively and Negatively Regulate the Self-Renewing Proliferation of Immature HPCs

For the purpose of studying hematological diseases involving complex lineages of cells, hematopoietic differentiation using patient-derived iPS cells has already proved to be powerful in the analysis of various diseases. However, in terms of sourcing for the expansion of its use to larger scale studies, the time-consuming and inefficient differentiation process still presents a significant obstacle. In this context, the development of a method that allows the proliferation of progenitor cells (HPCs) which maintains their ability to differentiate into multiple lineages is an important issue. In vivo, immature multipotent hematopoietic stem cells (HSCs) reside in the bone marrow, where intracellular signals via phosphorylation cascades co-ordinately regulate the expression pattern of transcription factors (TFs), leading to fate decisions within the microenvironment between self-renewal and one-way differentiation into HPCs. In contrast, iPS cell-derived hematopoietic differentiation does not involve HSCs and the microenvironment differs from that of bona fide cells. Therefore, the signal combinations required to amplify iPS cell-derived HPCs without further differentiation should naturally differ from those observed in HSCs in vivo. Based on this assumption, we constructed a CRISPRa and CRISPRi screen for phosphorylation-related genes using iPS cell-derived differentiation to search for factors that enable long-term maintenance of multipotent HPCs. First, Doxycycline-inducible dCas9-VPR (CRISPRa) or dCas9-KRAB-MecP2 (CRISPRi) expression cassettes were introduced into human iPS cells before, and then acted in combination with the phosphorylation-focused sgRNA libraries at the time when HPCs begin to be produced from mesodermal progenitors during their hematopoietic differentiation. Then, to identify the sgRNAs of interest with single-cell accuracy, colony formation assays were performed by isolating HPCs one by one by single-cell sorting. 2 weeks later, cells were collected only from wells that had formed CFU-Mix. Amplicon-sequences were performed using genomic DNA extracted from those cells and the sgRNAs that significantly contributed to the phenotype were identified by calculating the difference in enrichment with control sgRNAs. As a result, the CRISPRa screen identified a group of intracellular signals related with eight candidate genes known to be specifically activated in immature cells in the normal hematopoiesis. On the other hand, the results of CRISPRi showed that suppression of necroptosis-related signalling probably promotes long-term culture of HPCs in the stroma-free, artificial culture circumstances. Our results showed the potential to obtain multipotent HPCs from iPS cells over a long period of time and in large quantities, simply by adjusting the intracellular signals without artificial adding or removing of individual genes.

Depletion of Bone Marrow Hematopoietic Cells in Ebolavirus-Infected Rhesus Macaques: A Possible Cause of Hematologic Abnormalities in Ebolavirus Disease

The pathophysiology of long-recognized hematologic abnormalities in Ebolavirus (EBOV) disease (EVD) is unknown. From limited human sampling (of peripheral blood), it has been postulated that emergency hematopoiesis plays a role in severe EVD, but the systematic characterization of the bone marrow (BM) has not occurred in human disease or in nonhuman primate models. In a lethal rhesus macaque model of EVD, 18 sternal BM samples exposed to the Kikwit strain of EBOV were compared to those from uninfected controls (n = 3). Immunohistochemistry, RNAscope in situ hybridization, transmission electron microscopy, and confocal microscopy showed that EBOV infects BM monocytes/macrophages and megakaryocytes. EBOV exposure was associated with severe BM hypocellularity, including depletion of myeloid, erythroid, and megakaryocyte hematopoietic cells. These depletions were negatively correlated with cell proliferation (Ki67 expression) and were not associated with BM apoptosis during disease progression. In EBOV-infected rhesus macaques with terminal disease, BM showed marked hemophagocytosis, megakaryocyte emperipolesis, and the release of immature hematopoietic cells into the sinusoids. Collectively, these data demonstrate not only direct EBOV infection of BM monocytes/macrophages and megakaryocytes but also that disease progression is associated with hematopoietic failure, notably in peripheral cytopenia. These findings inform current pathophysiologic unknowns and suggest a crucial role for BM dysfunction and/or failure, including emergency hematopoiesis, as part of the natural history of severe human disease.

Human umbilical cord blood-derived mesenchymal stem cells restored hematopoiesis by improving radiation induced bone marrow niche remodeling in rats

BackgroundFunctional hematopoiesis is governed by the bone marrow (BM) niche, which is compromised by radiotherapy, leading to radiation induced BM failure. The aim of this study was to demonstrate the radiation induced pathological remodeling of the niche and the efficacy of human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) in restoring hematopoiesis via improvement of the niche. MethodsThirty male Wistar rats were equally assigned to three groups: control (CON), irradiated (IR), and IR+hUCB‐MSCs. Biochemical, histopathological and immunohistochemical analyses were performed to detect collagen type III and IV, Aquaporin 1+ sinusoidal endothelial cells and immature hematopoietic cells, CD11c+ dendritic cells, Iba1+ macrophages, CD9+ megakaryocytes, Sca-1+, cKit+, CD133 and N-cadherin+ hematopoietic stem and progenitor cells, CD20+, Gr1+ mature hematopoietic cells, in addition to ki67+ proliferation, Bcl-2+ anti-apoptotic, caspase-3+ apoptotic, TNF-α+ inflammatory cells. Histoplanimetry data were statistically analyzed using the one-way analysis of variance followed by the post hoc Duncan’s test. Moreover, Pearson’s correlation was used to assess the correlation between various parameters. ResultsIn comparison to the IR group, the IR+hUCB-MSCs group showed restored cell populations and extracellular collagen components of the BM niche with significant increase in hematopoietic stem, progenitor, mature and proliferating cells, and a considerable decrease in apoptotic and inflammatory cells. Furthermore, highly significant correlations between BM niche and blood biochemical, histopathological, and immunohistochemical parameters were observed. ConclusionhUCB-MSCs restored functional hematopoiesis through amelioration of the BM niche components via reduction of oxidative stress, DNA damage, inflammation, and apoptosis with upregulation of cellular proliferation.

Equivalents of the neutrophil-to-lymphocyte ratio of circulating pool of stem and immature hematopoietic cells for assessing liver transplant status

Objective: to study the applicability of the neutrophil-to-lymphocyte ratio (NLR) for monitoring recipient status and for possible minimization of maintenance immunosuppression in the long-term period after liver transplantation (LT).Materials and methods. Blood samples of 19 recipients with satisfactory graft function were examined by flow cytofluorometry at various time periods after LT using hematopoietic stem cell markers CD133, their CD31 derivatives, and alpha-fetoprotein (AFP), compared with the conventional NLR.Results. The use of NLR equivalents with CD133 and CD31 to assess liver transplant status is due to their high representation in liver tissue. Their values change in the long-term posttransplant period (from 1.5 to 6–7 years following LT) ≈20-fold and in different directions, but only when measuring their commissural to the liver cell fractions bearing the AFP marker.Conclusion. In contrast to the conventional NLR, maintenance of the lowest level of CD31 AFP, an NLR «equivalent», achieved at 1.5 years after LT, can be considered a criterion for the success of immunosuppressive therapy in the long-term post-LT period. The developed technique can be used to decide on whether to reduce or discontinue medication-assisted prophylaxis of graft rejection.

Increased Bone Marrow Uptake and Accumulation of Very-Late Antigen-4 Targeted Lipid Nanoparticles.

Lipid nanoparticles (LNPs) have evolved rapidly as promising delivery systems for oligonucleotides, including siRNAs. However, current clinical LNP formulations show high liver accumulation after systemic administration, which is unfavorable for the treatment of extrahepatic diseases, such as hematological disorders. Here we describe the specific targeting of LNPs to hematopoietic progenitor cells in the bone marrow. Functionalization of the LNPs with a modified Leu-Asp-Val tripeptide, a specific ligand for the very-late antigen 4 resulted in an improved uptake and functional siRNA delivery in patient-derived leukemia cells when compared to their non-targeted counterparts. Moreover, surface-modified LNPs displayed significantly improved bone-marrow accumulation and retention. These were associated with increased LNP uptake by immature hematopoietic progenitor cells, also suggesting similarly improved uptake by leukemic stem cells. In summary, we describe an LNP formulation that successfully targets the bone marrow including leukemic stem cells. Our results thereby support the further development of LNPs for targeted therapeutic interventions for leukemia and other hematological disorders.

Timing and regulation of hematopoietic stem cell colonization of the human fetal bone marrow by endothelial and CAR stromal cells during pregnancy

The bone marrow (BM) contains the major reservoir of immature and maturing hematopoietic and immune cells throughout adult life while also harboring most hematopoietic stem and progenitor cells (HSPCs). BM-retained hematopoietic stem cells (HSCs) are mostly maintained in a quiescent, non-motile mode. A small fraction of BM-retained HSPC daily proliferate, differentiate, and migrate to the circulation, to replenish the blood with new immature and maturing blood and (all) immune (both myeloid and lymphoid) cells with a finite life span.1 The anti-coagulation and anti-inflammatory receptor EPCR is also functionally expressed by primitive BM-retained HSCs which are endowed with the highest competitive long-term repopulation potential (LT-HSC). Only BM-retained, quiescent EPCR-positive LT-HSCs are protected from DNA damaging insults including clinical chemotherapy and radiation treatments. Primitive EPCR-positive LT-HSC chemotherapy resistance requires the CXCL12-CXCR4 axis that also regulates HSC quiescence, cell cycle, and directional migration as well as the aPC/EPCR/PAR1 axis.2,3 The chemokine CXCL12 is highly expressed by many BM endothelial and stromal cells types including HSC niche supporting osteoprogenitor cells termed CXCL12 abundant reticular cells (CAR cells), while primitive EPCR-positive fetal liver and adult BM HSC functionally express its major receptor CXCR4.4 Most functional HSC studies involve mice experimental preclinical models as well as results obtained from clinical BM transplantation protocols. During fetal development, HSCs migrate from the fetal liver to the fetal BM and spleen for their lodgment and repopulation. Stem cell homing to the BM during development and in functional experimental transplantation assays including with human HSC in transplanted immune deficient mice is CXCL12/CXCR4-dependent.5,6 The precise timepoint of HSC colonization of the human fetal BM and spleen during pregnancy, as well as the identity of BM stromal niche cells and ligand–receptor interactions which regulate human BM HSC lodgment during fetal development are poorly understood. The group of Zheng et al7 have recently reported on the emergence of HSC in the human fetal BM and spleen and their landscape microenvironment during pregnancy by single-cell RNAseq analysis. This work revealed that functional, human fetal LT-HSC as assayed in transplanted immune deficient mice do not emerge before week 12 post conception. By careful examination of various types of fetal BM stromal and endothelial cells during weeks 10–14 post conception and their transcriptome, this study revealed that fetal BM CAR-like cells endowed with the high levels of CXCL12 production together with fetal endothelial cells which also highly express CXCL12 are the major LT-HSC niche supporting cells. Interestingly, the human fetal spleen was not repopulated with functional human LT-HSC before week 14 post conception, revealing that fetal BM is superior in providing critical signals for LT-HSC homing and lodgment over the fetal spleen. Importantly, self-renewing human CD146 positive osteoprogenitor CAR-like cells were detected in adult human BM and were found to provide a niche for adult human LT-HSC as well as in a miniature human bone implanted in immune deficient mice.8 In conclusion, this study provides evidence for the first functional fetal BM LT-HSC as well as their BM supporting microenvironment and niches.

LSD1 inhibition modulates transcription factor networks in myeloid malignancies.

Acute Myeloid Leukemia (AML) is a type of cancer of the blood system that is characterized by an accumulation of immature hematopoietic cells in the bone marrow and blood. Its pathogenesis is characterized by an increase in self-renewal and block in differentiation in hematopoietic stem and progenitor cells. Underlying its pathogenesis is the acquisition of mutations in these cells. As there are many different mutations found in AML that can occur in different combinations the disease is very heterogeneous. There has been some progress in the treatment of AML through the introduction of targeted therapies and a broader application of the stem cell transplantation in its treatment. However, many mutations found in AML are still lacking defined interventions. These are in particular mutations and dysregulation in important myeloid transcription factors and epigenetic regulators that also play a crucial role in normal hematopoietic differentiation. While a direct targeting of the partial loss-of-function or change in function observed in these factors is very difficult to imagine, recent data suggests that the inhibition of LSD1, an important epigenetic regulator, can modulate interactions in the network of myeloid transcription factors and restore differentiation in AML. Interestingly, the impact of LSD1 inhibition in this regard is quite different between normal and malignant hematopoiesis. The effect of LSD1 inhibition involves transcription factors that directly interact with LSD1 such as GFI1 and GFI1B, but also transcription factors that bind to enhancers that are modulated by LSD1 such as PU.1 and C/EBPα as well as transcription factors that are regulated downstream of LSD1 such as IRF8. In this review, we are summarizing the current literature on the impact of LSD1 modulation in normal and malignant hematopoietic cells and the current knowledge how the involved transcription factor networks are altered. We are also exploring how these modulation of transcription factors play into the rational selection of combination partners with LSD1 inhibitors, which is an intense area of clinical investigation.

Diagnosis, prophylaxis and treatment of oral complications in children with acute lymphoblastic leukemia. A narrative review

Acute lymphoblastic leukemia (ALL) is a malignant condition defined by the proliferation of immature hematopoietic bone marrow cells, which replace the normal bone marrow elements. Furthermore, immature cells will accumulate in various areas of the body. Chemotherapeutic drugs are cytotoxic and have unwanted effects on normal cells. This toxicity is acute, or it may be chronic. Approximately 60% of survivors of ALL will suffer from one or more health problems or adverse side effects of this treatment. Oral complications are a frequent consequence of oncology therapy in pediatric patients. Xerostomia, carious lesions, gingivitis, tumors of the oral cavity, bone deformities of the jaw, temporomandibular joint disorders, trismus, and infections due to abundant microbial flora are common in these children. The risk of dental anomalies (dental or root agenesis, root malformations, enamel defects, dental hypoplasia) is higher in those who started treatment before permanent teeth erupt. All children are at risk of developing oral lesions due to poor hygiene, but children who have survived cancer are at even higher risk. The survival rate of children diagnosed with ALL has significantly increased thanks to remarkable advances in chemotherapy. Therefore, it is important that these patients are constantly monitored by the dentist to treat complications arising from the treatments in a timely manner. In children diagnosed with ALL, oral health is of great importance as the oral environment changes as a side effect of hematological disorders and their treatments. Treatment of carious lesions, periodontal problems and lesions of the oral mucosa must be done before starting chemotherapy by cleaning the oral cavity to eliminate any possible risk of infection. The participation of the dentist is important in improving the preventive protocols and oral examination of the pathological processes that occur in children with ALL. Interdisciplinary collaboration between physicians is essential during the treatment of pediatric cancer patients.

DIACC2010, Sole-in-Class Selective Inhibitor of Kinesin KIF20A, Has Potent Preclinical Efficacy in Acute Myeloid Leukemia

Context: Despite recent therapeutic advances in Acute Myeloid Leukemia (AML) and the decent proportion of complete remission after induction chemotherapy, relapse rate is still high and long-term survival does not exceed 20%. The therapeutic arsenal of AML could benefit from differentiated strategies. Mitotic kinesins are essential regulators of cancer cell replication and migration. The microtubule associated motor protein KIF20A (also called MKlp2), a member of the kinesin-6 family, plays an essential role during cytokinesis and is also involved in the fission of RAB6-positive vesicles from Golgi membrane, therefore contributing to intracellular vesicular trafficking. KIF20A plays a critical role in the development and progression of many cancers, and its high expression is associated with disease progression and poor survival outcome. More specifically, immature hematopoietic cells, exhibit high KIF20A expression, whereas mature peripheral blood cells do not. Herein we describe the preclinical anti-leukemic efficacy of DIACC2010, sole-in-class selective KIF20A inhibitor. Methods: We used cell lines (U-937, KG-1, CEM, KASUMI, SKM-1, MOLM-14, HL-60, MV-4-11, THP1) provided by ATCC and a Cell line-Derived Xenograft models to demonstrate the anti-leukemic activity od DIAC2010. Briefly, NOD.Cg-Prkdc scid/J (NSG) mice were obtained from Charles River France and maintained under specific pathogen-free conditions. Animals were injected via tail vein with 0.2 106 viable human leukemia cells. Mice were treated with 0.9% NaCl or Diacc2010 (200 mg/kg IV; once a week). Daily monitoring of mice for symptoms of disease determined the time of killing for animals with signs of distress. In addition, primary AML samples were obtained after informed consent. Cells were transplanted to NSG mice, collected after the 3rd transplantation and drug tests were performed ex vivo. All animal experiments were performed in compliance with the laws and in agreement with the French Guidelines for animal handling and approved by animal ethics committees (Agreement no. APAFIS#6743). Golgi imaging was performed using anti-GM130 antibody. Cell cycle, Caspase-Glo 3/7 and Annexin V/PI tests were performed according to the manufacturer's protocols. Results: DIACC2010 demonstrated potent and consistent cytotoxic activity in vitro against a panel of 9 human AML cell lines, with median IC50 of 40 nM (range 10-77 nM). In the same experimental conditions, cytarabine (CYTA) had median IC50 of 207 nM (range 4-1580 nM). DIACC2010 was subsequently evaluated in vivo in xenograft models of AML cell lines. DIACC2010 significantly improved overall survival as compared to CYTA in all models, with dose-dependent efficacy as shown in Figure A ( MOLM14 cells. Next, DIACC2010 cytotoxic activity was evaluated in vitro on 8 primary AML patient samples, IC50 ranged from 57 nM to 728 nM across the tested samples. DIACC2010 antitumoral activity is currently evaluated in Patients Derived Xenograft model. In parallel, no in vitro toxicity of DIACC2010 was observed on human normal cells such as peripheral blood mononuclear cells (PBMC) and primary hepatocytes (IC50 >50 µM). On a mechanistic standpoint, DIACC2010-exposed AML cells displayed characteristic Golgi scattering, observed within 1 hour of treatment and majoring at 6 hours confirming target engagement (Figure B). In parallel, we observed a strong block in cell cycle in G2/M after 6 hours of treatment (45% of cells versus 21% in the control condition). This was associated with a high caspase activation that occurred mainly after 24 hours of treatment. This cascade led to cell death assessed by annexin V/PI between 24 and 48 hours after treatment initiation. Nevertheless, caspase inhibitors ZVAD-fmk and Emricasan partially rescued DIACC2010 cell death, although these inhibitors significantly rescued caspase activation induced by Venetoclax, a BCL-2 inhibitor triggering intrinsic apoptosis commonly used in AML treatment. We ruled out necroptosis and ferroptosis as cell death pathways triggered by DIACC2010 using pharmacological inhibitors. These data strongly suggests that DIACC2010 induces a non-redundant cell death pathway that could be advantageously used to kill resistant AML cells. Conclusion: Altogether, these results confirm the relevance of KIF20A-directed therapeutic approaches and support the development of DIACC2010 for the treatment of AML. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Mlkl Mediates Age-Related Attrition of Hematopoietic Stem Cells and Ineffective Hematopoiesis in Myelodysplastic Syndrome

Aging causes anemia, perturbed immunity, and increased propensity to age-related hematological diseases such as myelodysplastic syndrome (MDS). Age-dependent functional decline in hematopoietic stem cells (HSCs), which is accompanied by morphological and functional changes in their organelles as well as sterile inflammation in the bone marrow (BM), is one of the main reasons why the hematopoietic system ages. However, molecular mechanisms by which HSCs alter their function during aging remain unclear. Necroptosis is a form of programmed necrosis that underlies sterile inflammation and can take place upon exposure to inflammatory stimuli in apoptosis-resistant cells including HSCs. MLKL, the effector molecule of necroptosis that can form channel-like structure on lipid bilayers upon activation, not only induces cell death but also injures membrane-bound organelles such as mitochondria, though its significance in HSC aging remains elusive. Our quantitative RT-PCR revealed abundant expression of MLKL in murine BM immature hematopoietic cells including HSCs (Lin-/cKit+/Sca1+/Flk2-/CD48-/CD150+), which was validated by reanalysis of the published proteome datasets (Zaro et al., eLife 2020). Using FRET-based necroptosis reporter mice and Western blotting analysis, we found that while MLKL was inactivated at the steady state, it was readily activated in HSCs upon poly I:C injection. To study the effect of MLKL activation on HSCs, we assessed cell death of HSCs isolated from poly I:C-treated MLKL-deficient (Mlkl-/-) mice. Unexpectedly, Annexin V/DAPI staining revealed no changes in the frequencies of dying and dead cells between wild-type (WT) and Mlkl-/- HSCs. Nonetheless, PVA-based HSC culture revealed that the growth suppression observed in poly I:C-treated WT HSCs largely depended on MLKL (p=0.0003). Furthermore, HSC transplantation assay revealed that reduced engraftment and skewed myeloid differentiation were observed in poly I:C-treated WT HSCs but largely alleviated by MLKL deficiency (p=0.011). These results indicate that inflammation-induced MLKL activation does not cause HSC death but impairs their growth and lymphopoietic potential. To assess the relevance of MLKL activation in HSC aging, we treated WT and Mlkl-/- mice with serial rounds of 5-FU treatment, which were shown to induce age-related alterations in HSCs (Beerman et al., Cell Stem Cell 2013). Of note, we observed marked attenuation of age-related features such as myeloid-skewed hematopoiesis and expansion of phenotypic HSCs in 5-FU-treated Mlkl-/- mice (p=0.0051). HSC transplantation experiments further revealed that MLKL deficiency alleviated 5-FU-induced decrease in engraftment and lymphopoietic potential of HSCs (p=0.013). Indeed, analysis of 18-month-old WT and Mlkl-/- mice demonstrated that MLKL deficiency largely rescued myeloid-skewed hematopoiesis and loss of BM CLPs (Lin-/cKitlo/Sca1lo/Flk2+/IL-7Rα+) (p=0.0047), and serial HSC transplantation revealed better self-renewal and lymphopoietic potential of aged Mlkl-/- HSCs (p=0.0055). Importantly, multiplex ELISA assay using BM supernatants revealed no changes in concentrations of inflammatory cytokines, indicating that the observed effects in Mlkl-/- HSCs were likely caused by cell intrinsic changes. In line with this, electron microscopy analysis revealed that age-related changes in mitochondria, such as elongated and swollen structure with disorganized cristae, were significantly attenuated in aged Mlkl-/- HSCs (p<0.0001). JC-1 dye staining further revealed that age-related accumulation of damaged mitochondria was significantly attenuated in Mlkl-/- HSCs (p=0.049). Finally, to evaluate the effect of MLKL on development of age-related hematological disease, we established MDS mouse models by transplantation of RUNX1S291fs-transduced WT and Mlkl-/- HSCs. We found that WT MDS mice mainly died of severe anemia due to ineffective hematopoiesis, whereas Mlkl-/- MDS mice showed milder anemia and better overall survival (p=0.044), indicating that MLKL promotes ineffective hematopoiesis in MDS. Taken together, our results identify MLKL as a key mediator of age-related functional decline in HSCs and ineffective hematopoiesis in MDS. They also suggest that pharmacological inhibition of MLKL activity is a promising strategy to better control aging of the hematopoietic system and age-related hematological disease.

Ectopic Viral Integration Site-1 Promotes NRAS/ERK Pathway through Epigenetic Silencing of microRNA-124 in AML

BACKGROUNDː Acute myeloid leukemia (AML) is an aggressive hematological malignancy characterized by genetic mutations that promote proliferation of myeloid progenitors and prevent their differentiation. Over-expression of Ectopic Viral Integration site-1(EVI-1) is related to the poor prognosis in myeloid leukemia, but the underlying mechanism remains unclear. METHODSː Using qRT-PCR and western blotting, we quantified expressions of EVI-1, NRAS and ERK/p-ERK in leukemia cell lines and PBMCs. Using WTS-8 and cell cycle analysis, we further investigated whether downregulation of EVI-1 by siRNA can inhibit cell proliferation. Microscopic observation of peripheral blood cells from EVI-1 transgenic zebrafish and WT control were analyzed by Wright Giemsa staining. Using miR-seq, qPCR, dual-luciferase reporter and coimmunoprecipitation assays, we revealed the relationship between EVI-1, miR-124 and NRAS. RESULTSː EVI-1 was highly expressed in both primary AML and leukemia cell lines (THP-1 and K562). In a transgenic zebrafish model, EVI-1 mediated higher mortality and induced immature hematopoietic cells in the blood circulation, suggesting its oncogenic role. Furthermore, our results suggested that EVI-1 upregulated NRAS expression, thereby activating the RAS/ERK pathway through epigenetic silencing of a potent NRAS suppressor, miR-124. In this study, we found that EVI1 physically interacts with Dnmt3a to form a protein complex that targets and binds to regulatory elements of miR-124. CONCLUSIONSː Overall, the current findings demonstrate that EVI-1 overexpression converges on the regulation of miR-124 promoter methylation and activation of the RAS/ERK pathway in AML carcinogenesis, and suggest EVI-1 and/or miR-124 as therapeutic targets for this dismal disease. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal