Hematopoietic Precursor Cells Research Articles

Estrogen deficiency in rats alters the expression and localization of redox proteins in the bone and bone marrow

Imbalances in bone formation/bone resorption ratio can occur under diverse conditions and diseases leading to an alteration of bone mineral density and strength. One of the most dramatic changes altering bone’s microarchitecture occurs under estrogen deficient conditions in humans and in other mammals. For instance, estrogen deficiency during menopause in women induces the formation and accumulation of reactive oxygen species (ROS) and a dysregulation of redox signaling. The bone and bone marrow are especially susceptible to the effects of oxidative stress and ROS. In tissues like the bone, cells are equipped with important defense systems to maintain homeostasis and to survive. Redox proteins of the thioredoxin family represent the most important protective system in cells that act against ROS and repair their intracellular effects. Redox proteins of the thioredoxin family comprise thioredoxins (Trxs), glutaredoxins (Grxs), and peroxiredoxins (Prxs). From all these protein classes, Prxs are the only ones that work as classical ROS scavengers being able to reduce hydrogen peroxide (H2O2), peroxynitrites and organic peroxides. Prxs are localized in different cellular compartments such as mitochondria (Prx3), cytosol and nucleus (Prx1 and Prx2), endoplasmic reticulum (Prx4), and peroxisomes (Prx5). We hypothesized that changes in estrogen levels may alter the expression and localization of Prxs in the bone and bone marrow. In this study, we analyzed the cellular localization and expression patterns of Prxs in the bone and bone marrow of rats as well as the effects of estrogens on their expression levels. We compared sham, ovariectomized, as well as ovariectomized/estrogen-replaced female rats of the same age. Western blot and immunohistochemistry revealed that all peroxiredoxins were abundantly expressed in different cell types of the bone and bone marrow such as osteocytes, chondrocytes, osteoclasts, bone lining cells, osteoblasts, and hematopoietic precursor cells. After ovariectomy, redox protein levels were significantly reduced in most cell types of the bone and bone marrow compartment. In addition to this, estrogen deficient animals showed increased levels of Prx1 in bone marrow supernatant suggesting a major redox dysregulation in this compartment. Estrogen replacement restored the levels of most redox proteins in the bone and bone marrow compartment and reduced the amount of extracellular Prx1. Based on these results, we conclude that estrogen deficiency negatively affects the ability of bone and bone marrow cells to maintain redox proteins at physiological levels. These results also reveal the widespread effects that are mediated by estrogen affecting different protein systems and tissues in the body. This work was funded by a Seed Discovery Grant (grant #3670920201102 to JG) from Long Island University. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

HES6 knockdown in human hematopoietic precursor cells reduces their in vivo engraftment potential and their capacity to differentiate into erythroid cells, B cells, T cells and plasmacytoid dendritic cells.

Hematopoiesis is driven by molecular mechanisms that induce differentiation and proliferation of hematopoietic stem cells and their progeny. This involves the activity of various transcription factors, such as members of the Hairy/Enhancer of Split (HES) family, and important roles for both HES1 and HES4 have been shown in normal and malignant hematopoiesis. Here, we investigated the role of HES6 in human hematopoiesis using in vitro and in vivo models. Using bulk and scRNA-seq data, we show that HES6 is expressed during erythroid/megakaryocyte and pDC development, as well as in multipotent precursors and at specific stages of T- and B-cell development following preBCR and preTCR signalling, respectively. Consistently, knockdown of HES6 in cord blood-derived hematopoietic precursors in well-defined in vitro differentiation assays resulted in reduced differentiation of human hematopoietic precursors towards megakaryocytes, erythrocytes, pDCs, Band T-cells. In addition, HES6 knockdown HSPCs displayed reduced colony forming unit capacity in vitro and impaired potential to reconstitute hematopoiesis in vivo in a competitive transplantation assay. We demonstrate that loss of HES6 expression impacts cell cycle progression during erythroid differentiation and provide evidence for potential downstream target genes that impact these perturbations. Thus, our study uncovers new insights for a role of HES6 in human hematopoiesis.

Abstract WP327: Altered Bone Marrow Microenvironment Impacts Endothelial Precursor Cell Development and Egress in Mice With Type 2 Diabetes

Endothelial precursor cells (EPCs) are vital contributors to angiogenesis, a crucial process in tissue repair and functional recovery after ischemic stroke. However, in the context of Type 2 Diabetes Mellitus (T2DM), angiogenesis is compromised, particularly after ischemic stroke. The mechanisms underlying the impacts of T2DM on EPC dynamics in the bone marrow (BM), as well as its repercussions on functional recovery post-stroke, remain incompletely understood. We found that the EPC populations were significantly diminished in the BM of diabetic mice compared to control mice prior to stroke by immunofluorescence staining (IF). Interestingly, the number of EPC was not significantly changed in the BM of diabetic mice after stroke, in contrast to the reduction seen in the control mice, suggesting an inherent impairment of EPC production and migration in the diabetic BM milieu. Consistent with this finding, VEGF, a critical factor for EPC proliferation and migration, was found to be increased in the BM in the control mice but reduced in the diabetic mice after ischemic stroke. There was also an overall reduction of CD115 and Ly6G expression in the BM of diabetic mice, indicative of an altered microenvironment for hematopoiesis that could further affect EPC dynamics. Single-cell RNA sequencing of cells revealed the presence of hematopoietic precursor cells (HPC), monocytes, granulocytes, and macrophages among the clustered cell populations. Enrichment analysis unveiled pathways associated with mitotic DNA replication, myeloid cell differentiation, and leukocyte migration among the differentially expressed genes in HPC between diabetic and control BM. Strikingly, genes linked to the angiogenesis and endothelial cell migration were also down regulated in T2DM BM, potentially contributing to the impaired angiogenic response observed in T2DM after stroke. These findings underscore the intricate interplay among T2DM, EPC function, angiogenesis, and post-stroke recovery. The identification of disrupted pathways in the BM environment provides valuable insights into the compromised angiogenic response observed in diabetes and novel targets for therapeutic interventions in improving stroke outcomes in patients with T2DM.

Parbendazole as a promising drug for inducing differentiation of acute myeloid leukemia cells with various subtypes

Acute myeloid leukemia (AML) is a malignancy characterized by differentiation arrest of hematopoietic precursor cells. Differentiation therapy is effective for patients with acute promyelocytic leukemia; however, only a few effective differentiation therapies have been established for patients with other AML subtypes. In this study, seven benzimidazole anthelmintics were examined to determine the effects of differentiation on AML cells. The expression of monocyte markers (CD11b and CD14) was elevated after treatment with most benzimidazole anthelmintics. Among these drugs, parbendazole (PBZ) induced AML cell differentiation at low concentration. PBZ induced the monocyte marker expression, KLF4/DPYSL2A gene expression, and apoptosis for 21 AML cell lines with various subtypes and a primary AML sample. Finally, an in vivo analysis using an AML patient-derived xenograft mouse model showed a significant decrease in the chimerism level and prolonged survival in PBZ-treated mice. These findings could lead to a more effective differentiation therapy for AML.

Gemtuzumab Ozogamicin in Acute Myeloid Leukemia: Efficacy, Toxicity, and Resistance Mechanisms-A Systematic Review.

Acute myeloid leukemia (AML) is a diverse group of leukemias characterized by the uncontrolled proliferation of clonal neoplastic hematopoietic precursor cells with chromosomal rearrangements and multiple gene mutations and the impairment of normal hematopoiesis. Current efforts to improve AML outcomes have focused on developing targeted therapies that may allow for improved antileukemic effects while reducing toxicity significantly. Gemtuzumab ozogamicin (GO) is one of the most thoroughly studied molecularly targeted therapies in adults. GO is a monoclonal antibody against CD33 IgG4 linked to the cytotoxic drug calicheamicin DMH. The use of GO as a chemotherapeutic agent is not generalized for all patients who suffer from AML, particularly for those whose health prevents them from using intensive conventional chemotherapy, in which case it can be used on its own, and those who have suffered a first relapse, where its combination with other chemotherapeutic agents is possible. This systematic review aimed to comprehensively evaluate GO, focusing on its molecular structure, mode of action, pharmacokinetics, recommended dosage, resistance mechanisms, and associated toxicities to provide valuable information on the potential benefits and risks associated with its clinical use. A systematic review of eight scientific articles from 2018 to 2023 was conducted using PRISMA analysis. The results showed that GO treatment activates proapoptotic pathways and induces double-strand breaks, initiating DNA repair mechanisms. Cells defective in DNA repair pathways are susceptible to GO cytotoxicity. GO has recommended doses for newly diagnosed CD33+ AML in combination or as a single agent. Depending on the treatment regimen and patient status, GO doses vary for induction, consolidation, and continuation cycles. Multidrug resistance (MDR) involving P-glycoprotein (P-gp) is associated with GO resistance. The overexpression of P-gp reduces GO cytotoxicity; inhibitors of P-gp can restore sensitivity. Mitochondrial pathway activation and survival signaling pathways are linked to GO resistance. Other resistance mechanisms include altered pharmacokinetics, reduced binding ability, and anti-apoptotic mechanisms. GO has limited extramedullary toxicity compared to other AML treatments and may cause hepatic veno-occlusive disease (HVOD). The incidence of hepatic HVOD after GO therapy is higher in patients with high tumor burden. Hematological side effects and hepatotoxicity are prominent, with thrombocytopenia and neutropenia observed. In conclusion, GO's reintroduction in 2017 followed a thorough FDA review considering its altered dose, dosing schedule, and target population. The drug's mechanism involves CD33 targeting and calicheamicin-induced DNA damage, leading to apoptosis and resistance mechanisms, including MDR and survival signaling, which impact treatment outcomes. Despite limited extramedullary toxicity, GO is associated with hematological side effects and hepatotoxicity.

Acute Leukemia in A Young Female Adult Presenting for Care in A Resource Constrained Setting

Background: Acute leukemia is a rare malignant disorder arising from an abnormal proliferation of hematopoietic precursor cells. With advances in the management of leukemia, patients of acute leukemia have high chances of achieving adequate remission and survival. However, due to late presentation, diagnosis and treatment, the disease has significantly contributed to high morbidity and mortality especially in resource limited settings. Method: A comprehensive history and physical examination was taken, coupled with literature review. Various rationale for each intervention and recommended interventions unavailable in our setting were noted. Results: Early diagnosis and treatment of leukemia and its complications is a major public health concern for which the general public needs to be educated on. In this case report, material and human resource constraints in our setting may contribute towards the patient’s poor clinical outcome. Conclusion and recommendations: Acute leukemia is a manageable disorder provided the availability of appropriate resources and interventions being implemented early. There is therefore need to equip our health facilities and personnel with resources towards early clinical detection, diagnosis and treatment which measures are essential for successful management and prevention of unwanted complications.

An Analytic Framework for the Prediction of Gvl Minor Histocompatibility Antigens to Prevent or Treat Post-Transplant Relapse

The therapeutic effect of allo-HSCT relies on T cell alloreactivity driven by genetic nonidentity between donor and recipient (D-R) pairs. In the setting of HLA-matched transplants, endogenous proteins in recipient cells differing from those of the donor due to genetic polymorphisms, can provide distinct HLA-binding peptides, serving as minor histocompatibility antigens (mHAgs). Hematopoietically-restricted mHAgs represent ideal targets to separate graft-versus-leukemia (GvL) from graft-versus-host-disease (GvHD) effects. To date,however, the quest for optimal GvL mHAgs has resulted in the identification of only a handful of targets, raising concerns on the clinical feasibility of their targeting. We hypothesized that systematic mHAg prediction from whole-exome sequencing (WES) of D-R pairs could help identifying novel candidate mHAgs with an acceptable safety profile for therapeutical targeting. We focused on AML/MDS, the most common indications for allo-HSCT in adults. To capture the transcriptional heterogeneity of malignant myeloid cells, we applied a single-cell based classifier to define genes expressed by leukemic cells in the Beat AML cohort, and then removed those expressed in non-hemopoietic tissues, per the GTEx RNA and protein database. Similarly, a broader set of pan-hematopoietic genes was defined starting from the expression profiles of 18 mature hematopoietic lineages and hematopoietic stem and precursor cells. Through this process, we identified 259 genes with preferential expression in AML and 615 broadly expressed in the hematopoietic compartment. We applied this analytical pipeline to WES from 220 HLA-matched D-R pairs transplanted at DFCI. To discover mHAgs with optimal GvL potential, we focused on the subgroup of 45 patients with long-term survival in the absence of both relapse and GvHD requiring systemic treatment (i.e. GvHD-free/relapse-free survival [GRFS]). For these patients experiencing purely GvL, we analyzed the individual repertoire of predicted GvL mHAgs, and found 86 SNPs recurring in ≥5 GRFS patients. These GvL SNPs were enriched in the subgroup of GRFS patients versus those without GRFS, suggesting that their overrepresentation was not dictated by high allelic frequency in the overall cohort. Using the median number of recurring GvL mHAgs in the GRFS subgroup (‘GRFS mHAgs‘) as a cutoff to stratify the entire study cohort, we found that a higher GRFS mHAg load was associated with a protective effect against relapse ( p=0.009) and conferred a benefit in 2-year overall survival ( p=0.03). Furthermore, HLA class I immunopeptidome analysis of 5 AML cell lines revealed evidence of presentation for 10 epitopes predicted from genes in the overall GvL filter, two of which were also part of the GRFS mHAg set. Given the limited size and lack of ethnic diversity of our discovery cohort (composed mainly of patients of European ancestry), we used genomic data from the 1000 Genomes project to estimate the feasibility of targeting our GRFS mHAg set in a broader population via in silico modeling of allo-HSCT. We simulated 2270 D-R pairs from 844 individuals across 5 populations of disparate ancestry (AFR, EAS, EUR, SAS, and AMR), mirroring a typical URD search in the NMDP registry. To gauge the feasibility of generating personalized mHAg-specific immunotherapy (i.e. cancer vaccines, adoptive cellular therapy) based on the GRFS mHAg set, we estimated that a 10-SNP design (i.e., the number of GRFS SNPs used for correlative analyses in our discovery cohort) would be possible for ~90% of the 1000 Genomes simulated cohort (EUR: 99%, EAS: 72%, SAS: 91%, AFR: 69%, AMR: 74%). Through a similar process, we calculated population coverage simulating a T cell-based approach targeting ≥1 epitope from the GRFS mHAg set. First, we identified 18 HLA alleles that could ensure ~99% coverage across diverse ethnic backgrounds. Second, for each HLA allele, the 3 most frequently predicted (and with highest agretopicity) epitopes from the GRFS SNP set were selected, defining a pool of 54 epitopes. We thus calculated that ≥1 epitopes could be potentially targeted for 81% of the simulated D-R pairs (EUR: 91%, EAS: 78%, SAS: 71%, AFR: 62%, and AMR:85%). Overall, we developed a robust approach to identify novel GvL mHAgs, which could form the basis for systematic personalized immunotherapy following allo-HSCT.

Can Emergency Hematopoiesis Due to Sepsis Lead to Clonal Hematopoiesis? Implications for Understanding the Pathogenesis of Clonal Evolution

O.O & T.K are co-first authors. Clonal (myelo) hematopoiesis of indeterminate potential (CHIP) arises from somatic mutations in myeloid genes, providing a proliferative and survival advantage to the affected “CHIP-initiating” early hematopoietic precursor cells. Understanding the conditions that lead to the emergence of detectable clones (early seeding of myeloid neoplasia-MN) is of great interest due to its clinical implications. Among CH facilitating states, aging has been extensively studied, but a clear distinction between the impact of chronological versus biological aging remains challenging. Additionally, other factors contributing to CHIP development may include chemotherapy, genetic predisposition, bone marrow failure states, or immunodeficiency. However, not all CHIP-promoting factors have been identified because they are subtle, pathophysiologically complex, and thus remain cryptic. We hypothesized that episodes of emergency hematopoiesis (EH), such as those caused by acute infections, could potentially result in acquisition of CHIP with repeated episodes of EH resulting in clonal expansion. If this hypothesis is confirmed, one could envision that both instances of significant hematopoietic expansion (high leukocytosis) and repeated cycles of EH could be at play in conjunction with chronological factors. For instance, the older an individual is, the greater is the number of EH episodes possible. To examine our theory, we prospectively collected blood samples from patients admitted at the intensive care unit (ICU) of Cleveland Clinic and Yale University. Inclusion criteria required a white blood cell (WBC) count ≥ 15K/uL. Patients with known or prior malignancies and those exposed to chemotherapy were excluded. For relevant comparisons, we assessed CH incidence in our cohort against a group of patients with solid tumors screened for CH (n=107) but without acute infections, along with historical CHIP cohorts. CH screening employed a targeted NGS myeloid gene panel with a reported VAF cut-off of 2%. A total of 78 individuals admitted to the ICU with possible infections and leukocytosis were enrolled in this study. The median age was 70 years (IQR: 61-77), with a M:F ratio of 1.7 (Table 1). Time from leukocytosis onset to sampling was 3 days (IQR: 1-5), the median WBC at ICU admission was 18 K/uL (IQR: 16-21), and the median absolute neutrophil count (ANC) was 16 K/uL (IQR: 12-20). Overall, 94 variants were identified including 16 (median VAF 50.1) variants of potentially germline origin, and 4 variants of unknown significance. We observed 74 CHIP-associated mutations in 37 (47%) patients at a median VAF of 14% (4-47%). The landscape of CHIP mutations included alterations in DNMT3A (20%), TET2 (12%), ASXL1 (12%), SMC1A, TP53, and PPM1D genes (6% each). Although higher rates of CHIP were expected in the cancer cohort, no difference was found between the two groups (40% vs. 47% in the ICU, p=0.3). DNMT3A, ASXL1 and PPM1D mutations were more frequently encountered in the cancer cohort (Table 1). The prevalence of CHIP in the ICU cohort was higher in older age groups: 30-39 (29%), 50-69 (49%), and ≥70 (51%), compared to the expected frequency in comparable age ranges (and median range) in a historical healthy population, which was 1%, 4%, and 10%, respectively (<0.00001; Fig.1). Notably, CHIP was more prevalent among smokers (57%; p=0.003). No significant differences were found when comparing CHIP + and CHIP - patients of the ICU cohort with regards to WBC (median 18K/uL both). Similarly, CHIP prevalence did not change according to the degree of leukocytosis in an analysis where patients were ranked by WBC (≥18K/uL at 44% vs. <18K/uL at 46%). When looking at the impact of CHIP on critical care outcomes, a logistic regression model accounting for age, gender, and mutation type revealed that the presence of DNMT3A mutations was associated with a higher risk of diagnosis of overt septic shock (log OR= 2.4, 95% CI 0.19-5.4, p<0.05). In summary, our prospective observational study has shown that sepsis is associated with preferential mutant clone expansion, primarily due to EH rather than the extent of leukocytosis and may be contributing to the acquisition of new clones. Whether such episodes result in permanent acquisition of new CHIP mutations with potential for MN progression will be the subject of longitudinal analysis of these samples, and findings will be presented at the ASH meeting.

Mertk-expressing microglia influence oligodendrogenesis and myelin modelling in the CNS

BackgroundMicroglia, an immune cell found exclusively within the CNS, initially develop from haematopoietic stem cell precursors in the yolk sac and colonise all regions of the CNS early in development. Microglia have been demonstrated to play an important role in the development of oligodendrocytes, the myelin producing cells in the CNS, as well as in myelination. Mertk is a receptor expressed on microglia that mediates immunoregulatory functions, including myelin efferocytosis.FindingsHere we demonstrate an unexpected role for Mertk-expressing microglia in both oligodendrogenesis and myelination. The selective depletion of Mertk from microglia resulted in reduced oligodendrocyte production in early development and the generation of pathological myelin. During demyelination, mice deficient in microglial Mertk had thinner myelin and showed signs of impaired OPC differentiation. We established that Mertk signalling inhibition impairs oligodendrocyte repopulation in Xenopus tadpoles following demyelination.ConclusionThese data highlight the importance of microglia in myelination and are the first to identify Mertk as a regulator of oligodendrogenesis and myelin ultrastructure.

Flow cytometric analysis of CD34+ CD38- cells; cell frequency and immunophenotype based on CD45RA expression pattern.

The CD34+ CD38- population in bone marrow includes hematopoietic stem/progenitor cells. Recently, in acute myeloid leukemia, the focus has shifted to flow cytometry analysis targeting CD34+ CD38- leukemic cells due to their effectiveness in minimal/measurable residual disease detection and prognosis prediction. Nevertheless, the immunophenotype and cell frequency of these cells in the bone marrow, in the absence of leukemic cells, remains unknown. We aimed to evaluate detailed characteristics of CD34+ CD38- cells in both normal and leukemic cells by flow cytometry. We compared the cell frequency and immunophenotype of the CD34+ CD38- fraction in the following groups: patients with idiopathic thrombocytopenic purpura and malignant lymphoma as controls (n = 17), post-treatment patients without abnormal blasts (n = 35), and patients with myeloid malignancies (n = 86). The comparison was based on the presence or absence of CD45RA expression, a marker commonly used to prospectively isolate lymphoid-primed cell populations within the CD34+ CD38- fraction. The CD34+ CD38- CD45RA+ cell population exhibited a significant expansion in bone marrow without leukemic cells 1 month after cord blood transplantation and in various type of myeloid malignancies, compared to the control group (p < 0.01). Continuous CD45RA expression and notable expansion of the CD34+ CD38- CD45RA- population were exclusively observed in myelodysplastic syndrome-related diseases. The CD34+ CD38- CD45RA+ population displayed frequent expression of various markers in both leukemic and non-leukemic cells, in contrast to the CD34+ CD38- CD45RA- population. The CD34+ CD38- fraction should be carefully evaluated considering the nature of normal hematopoietic precursor cells, their cell frequency and immunophenotype, including CD45RA expression pattern, for improving the accuracy of myeloid malignancy diagnosis.

CD33-TIM3 Dual CAR T Cells: Enhancing Specificity While Maintaining Efficacy Against AML

Background: The myeloid associated antigen CD33 is overexpressed in over 88% of Acute myeloid leukemia (AML) patients, making it a suitable target antigen for CAR T cell therapy across different genetic subtypes. Despite the development of clinical trial data, there remains a concern about on-target off-leukemia toxicity. To enhance specificity, we generated dual targeting CAR T cells that target CD33 and/or T-cell immunoglobulin and mucin-domain containing-3 (TIM3). The latter is known to be expressed on LSCs, but not on healthy hematopoietic precursor or stem cells. Notably, TIM3 is recognized for its inhibitory immunomodulatory role further enhancing the suitability of this target antigen. The objective of this research was to explore various CD33 and TIM3 dual-targeting CAR T cells approaches that enhance AML specificity and maintain high anti-AML activity. Methods: The hybridoma technology was used to generate antibodies against TIM3 immunized mice. Antibodies were screened for TIM3 specificity via ELISA and FACS. The anti-TIM3 single-chain variable fragment (scFv) DNA sequence was sequenced from hybridoma cells. Furthermore, the binding ability to TIM3 antigen (PDB ID: 5F71) was evaluated using AlphaFold. The CD33 scFv was derived from Gemtuzumab ozogamicin (clone: hP67.6). ScFv sequences and co-stimulation domains (CD28 or 4-1BB) were cloned into a pMP71 vector. Retrovirus for transduction was produced using the 293Vec-GALV and RD114 retroviral production system. In vitro co-culture assays of CAR T cells and target cells were performed to study the efficacy of CAR T cells. The cytotoxicity against wild-type and TIM3 transduced AML cell lines (THP-1 and OCI-AML3) was assessed by multiparameter flow cytometry (MPFC). The secretion of effector cytokines (IFN-γ, TNF, IL-2) was analyzed via CBA assays. In addition, avidity between CAR T cells and target cells was determined by Z-Movie analyzer. The different CAR constructs were screened for on-target off-tumor toxicity in colony forming unit assays (CFU) using isolated CD34 + hematopoietic stem and progenitor cells (HSPCs) from healthy doners after 14 days. In order to compare CAR constructs regarding long-term efficacy in antigen restimulation assays, CAR T cells were co-cultured with irradiated TIM3 transduced OCI-AML3 every 4 days at an E: T ratio of 1:1 for 24 days. Moreover, CAR T-cell proliferation, checkpoint marker expression and T-cell subset differentiation were analyzed via MPFC. Results: All dual CAR T cells (compound, split, tandem, pooled) were generated with high and robust transduction efficacy (Figure 1A). TIM3-dependent fratricide was not observed during CAR T cell manufacturing. Compared to single antigen targeting CAR T cells, dual CAR T cells showed enhanced cytotoxicity against AML cell lines and primary AML cells (Figure 1B). Moreover, we also observed a strong increase in the secretion of proinflammatory cytokines (IFN-γ and IL-2) and higher avidity of dual CAR T cells. Notably, split CAR T cells demonstrated greater specificity in cocultures directed against mono vs. dual-target antigen expression cell lines. In accordance with these observations, split CAR T cells did not exert on-target off-leukemia toxicity against healthy HSC in CFU assays. In the antigen restimulation assay, we observed that compound CAR T cells exhibited diminished expanding capacity and heightened expression of exhaustion markers in comparison to the other CAR T constructs. Conclusion: We developed multiple CD33 and TIM3 dual CAR T cells using both “AND” and “OR” gating strategies in this study. Our findings revealed that dual CAR T cells provided higher avidity and cytotoxicity compared to single targeting CAR T cells against dual antigen expressing target cells in vitro. Importantly, only the split CAR T constructs demonstrated specific killing of CD33 +TIM3 + cell lines and primary AML cells while sparing HSPCs. Prospectively, compound, tandem, pooled CD33 TIM3 specific CAR T cells might be helpful in a bridge to transplant setting, whereas split CAR T cells might provide a higher safety profile, thereby allowing a transplant independent approach. To advance our concepts, we are currently conducting in vivo experiments in an NSG mouse model.

The Molecular and Genetic Mechanisms of Inherited Bone Marrow Failure Syndromes: The Role of Inflammatory Cytokines in Their Pathogenesis.

Inherited bone marrow failure syndromes (IBMFSs) include Fanconi anemia, Diamond-Blackfan anemia, Shwachman-Diamond syndrome, dyskeratosis congenita, severe congenital neutropenia, and other rare entities such as GATA2 deficiency and SAMD9/9L mutations. The IBMFS monogenic disorders were first recognized by their phenotype. Exome sequencing has validated their classification, with clusters of gene mutations affecting DNA damage response (Fanconi anemia), ribosome structure (Diamond-Blackfan anemia), ribosome assembly (Shwachman-Diamond syndrome), or telomere maintenance/stability (dyskeratosis congenita). The pathogenetic mechanisms of IBMFSs remain to be characterized fully, but an overarching hypothesis states that different stresses elicit TP53-dependent growth arrest and apoptosis of hematopoietic stem, progenitor, and precursor cells. Here, we review the IBMFSs and propose a role for pro-inflammatory cytokines, such as TGF-β, IL-1β, and IFN-α, in mediating the cytopenias. We suggest a pathogenic role for cytokines in the transformation to myeloid neoplasia and hypothesize a role for anti-inflammatory therapies.

Phenotypic profiling of CD34+ cells by advanced flow cytometry improves diagnosis of juvenile myelomonocytic leukemia.

Diagnostic criteria for juvenile myelomonocytic leukemia (JMML) are currently well defined, however in some patients diagnosis still remains a challenge. Flow cytometry is a well established tool for diagnosis and follow-up of hematological malignancies, nevertheless it is not routinely used for JMML diagnosis. Herewith, we characterized the CD34+ hematopoietic precursor cells collected from 31 children with JMML using a combination of standardized EuroFlow antibody panels to assess the ability to discriminate JMML cells from normal/reactive bone marrow cell as controls (n=29) or from cells of children with other hematological diseases mimicking JMML (n=9). CD34+ precursors in JMML showed markedly reduced B-cell and erythroid-committed precursors compared to controls, whereas monocytic and CD7+ lymphoid precursors were significantly expanded. Moreover, aberrant immunophenotypes were consistently present in CD34+ precursors in JMML, while they were virtually absent in controls. Multivariate logistic regression analysis showed that combined assessment of the number of CD34+CD7+ lymphoid precursors and CD34+ aberrant precursors or erythroid precursors had a great potential in discriminating JMMLs versus controls. Importantly our scoring model allowed highly efficient discrimination of truly JMML versus patients with JMML-like diseases. In conclusion, we show for the first time that CD34+ precursors from JMML patients display a unique immunophenotypic profile which might contribute to a fast and accurate diagnosis of JMML worldwide by applying an easy to standardize single eight-color antibody combination.

An Overview of Targeted Therapies in Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is the most aggressive adult leukemia, characterized by clonal differentiation arrest of progenitor or precursor hematopoietic cells. Intense preclinical and clinical research has led to regulatory approval of several targeted therapeutics, administered either as single agents or as combination therapies. However, the majority of patients still face a poor prognosis and disease relapse frequently occurs due to selection of therapy-resistant clones. Hence, more effective novel therapies, most likely as innovative, rational combination therapies, are urgently needed. Chromosomal aberrations, gene mutations, and epigenetic alterations drive AML pathogenesis but concurrently provide vulnerabilities to specifically target leukemic cells. Other molecules, either aberrantly active and/or overexpressed in leukemic stem cells, may also be leveraged for therapeutic benefit. This concise review of targeted therapies for AML treatment, which are either approved or are being actively investigated in clinical trials or recent preclinical studies, provides a flavor of the direction of travel, but also highlights the current challenges in AML treatment.

Mapping the Immune Cell Microenvironment with Spatial Profiling in Muscle Tissue Injected with the Venom of Daboia russelii.

Pathological and inflammatory events in muscle after the injection of snake venoms vary in different regions of the affected tissue and at different time intervals. In order to study such heterogeneity in the immune cell microenvironment, a murine model of muscle necrosis based on the injection of the venom of Daboia russelii was used. Histological and immunohistochemical methods were utilized to identify areas in muscle tissue with a different extent of muscle cell damage, based on the presence of hypercontracted muscle cells, a landmark of necrosis, and on the immunostaining for desmin. A gradient of inflammatory cells (neutrophils and macrophages) was observed from heavily necrotic areas to less damaged and non-necrotic areas. GeoMx® Digital Spatial Profiler (NanoString, Seattle, WA, USA) was used for assessing the presence of markers of various immune cells by comparing high-desmin (nondamaged) and low-desmin (damaged) regions of muscle. Markers of monocytes, macrophages, M2 macrophages, dendritic cells, neutrophils, leukocyte adhesion and migration markers, and hematopoietic precursor cells showed higher levels in low-desmin regions, especially in samples collected 24 hr after venom injection, whereas several markers of lymphocytes did not. Moreover, apoptosis (BAD) and extracellular matrix (fibronectin) markers were also increased in low-desmin regions. Our findings reveal a hitherto-unknown picture of immune cell microheterogeneity in venom-injected muscle which greatly depends on the extent of muscle cell damage and the time lapse after venom injection.

Novel causative variants of VEXAS in UBA1 detected through whole genome transcriptome sequencing in a large cohort of hematological malignancies

UBA1 is an X-linked gene and encodes an ubiquitin-activating enzyme. Three somatic mutations altering the alternative start codon (M41) in UBA1 in hematopoietic precursor cells have recently been described, resulting in a syndrome of severe inflammation, cytopenias, and the presence of intracellular vacuoles in hematopoietic precursors - termed VEXAS syndrome, a predominantly male disease. Here we present a patient with clinical features of VEXAS who harbored two novel somatic variants in UBA1 (I894S and N606I). To better understand the clinical relevance and biological consequences of non-M41 (UBA1non-M41) variants, we analyzed the whole genome and transcriptome data of 4168 patients with hematological malignancies and detected an additional 16 UBA1non-M41 putative somatic variants with a clear sex-bias in patients with myeloid malignancies. Patients diagnosed with myeloid malignancies carrying UBA1non-M41 putative somatic variants either had vacuoles or immunodysregulatory symptoms. Analysis of the transcriptome confirmed neutrophil activation in VEXAS patients compared to healthy controls but did not result in a specific transcriptomic signature of UBA1M41 patients in comparison with MDS patients. In summary, we have described multiple putative novel UBA1non-M41 variants in patients with various hematological malignancies expanding the genomic spectrum of VEXAS syndrome.

Clinicogenomic associations in childhood Langerhans cell histiocytosis: an international cohort study.

Langerhans cell histiocytosis (LCH) is a rare neoplastic disorder caused by somatic genetic alterations in hematopoietic precursor cells differentiating into CD1a+/CD207+ histiocytes. LCH clinical manifestation is highly heterogeneous. BRAF and MAP2K1 mutations account for ∼80% of genetic driver alterations in neoplastic LCH cells. However, their clinical associations remain incompletely understood. Here, we present an international clinicogenomic study of childhood LCH, investigating 377 patients genotyped for at least BRAFV600E. MAPK pathway gene alterations were detected in 300 (79.6%) patients, including 191 (50.7%) with BRAFV600E, 54 with MAP2K1 mutations, 39 with BRAF exon 12 mutations, 13 with rare BRAF alterations, and 3 with ARAF or KRAS mutations. Our results confirm that BRAFV600E associates with lower age at diagnosis and higher prevalence of multisystem LCH, high-risk disease, and skin involvement. Furthermore, BRAFV600E appeared to correlate with a higher prevalence of central nervous system (CNS)-risk bone lesions. In contrast, MAP2K1 mutations associated with a higher prevalence of single-system (SS)-bone LCH, and BRAF exon 12 deletions seemed to correlate with more lung involvement. Although BRAFV600E correlated with reduced event-free survival in the overall cohort, neither BRAF nor MAP2K1 mutations associated with event-free survival when patients were stratified by disease extent. Thus, the correlation of BRAFV600E with inferior clinical outcome is (primarily) driven by its association with disease extents known for high rates of progression or relapse, including multisystem LCH. These findings advance our understanding of factors underlying the remarkable clinical heterogeneity of LCH but also question the independent prognostic value of lesional BRAFV600E status.

The Role of Stem Cell on Orthodontic Tooth Movement Induced-Alveolar Bone Remodeling

Alveolar bone tissue constantly undergoes remodeling through new bone formation and bone resorption. Osteoclasts originated from hematopoietic precursor cells and monocytes/macrophage lineage. In particular it will differentiate into mononuclear preosteoclasts and will merge into multinucleated osteoclast. Osteoblasts originated from undifferentiated mesenchymal stem cells. Osteoprogenitor cells evolved into preosteoblasts, and then into osteoblasts and osteocytes latter, which has the capability of bone mineralization and calcification. Orthodontic mechanical force responded directly by MSC to perform self-renewal and osteogenic differentiation, whereas HSC respond to mechanical force mediated by osteoblastic lineage cell in osteoclastic differentiation.

A case of sudden death due to pulmonary embolism of bone marrow origin, masked as acute drug toxicity: A rare case in a bewildering clinical scenario

Sudden death is unexpected death within 24 hours from the onset of symptoms with or without known preexisting conditions. Respiratory pathologies are the second most common cause of such deaths. 39 years old male patient with a history of mental illness for the last 10 years and on regular treatment with antipsychotic medications, suffered sudden deterioration of higher functions and was admitted to the Medical College, and Hospital, Kolkata with a provisional diagnosis of drug toxicity. He succumbed to his condition within 24 hours of admission and the dead body was referred to Kolkata Police Morgue for a medicolegal autopsy.Shows multi-system involvement in the form of Pulmonary Oedema, consolidation, broncho-pneumonic changes and subpleural petechiae, Cardiomegaly due to biventricular hypertrophy, Cerebral Oedema, Enlarged kidneys with loss of cortico-medullary differentiation and streaky cortico-medullary haemorrhages. Stomach findings were unremarkable.1) Toxicological Examination of blood, bile, vitreous and routine viscera was negative for any poison or pharmacological agents.2)Histopathological examination with routine H&amp;E staining showed Pulmonary oedema with lymphocytic infiltration and the presence of fat and hematopoietic precursor cells in pulmonary vessels, Interstitial Oedema and focal glomerulosclerosis and Periportal inflammation in the liver.

Paratesticular Extramedullary Hematopoiesis in Children.

Extramedullary hematopoiesis (EMH) is an uncommon occurrence, usually associated with hematologic disorders, but it rarely presents as an isolated finding. To determine the frequency, immunomorphologic features, and clinicopathologic background of EMH in orchiectomies from pediatric patients. All orchiectomy specimens removed from children from 2008 to 2020 in our institution were retrospectively reviewed. Biopsies and neoplasias were excluded. The EMH diagnosis was rendered when hematopoietic cell precursors were present. Immunohistochemical stainings were performed to characterize the hematopoietic components. Seventy-nine orchiectomies from 77 children (mean age, 5 years; range, 0-17 years) were included in our study. Forty-three patients (55.8%) underwent surgery for testicular atrophy, 30 (39.0%) for torsion, and 4 (5.2%) for intersex conditions. EMH was identified in 6 of 79 orchiectomies (7.6%), all performed for testicular torsion. All patients but one were newborns, and the remaining patient was 15 years old. No patient had evidence of a hematologic disorder. All EMH foci were in a background of reactive changes with a variable extension, either in the epididymis (4 cases) or in the deferens duct (2 cases). Immunostaining confirmed an association of myeloid (myeloperoxidase+) and erythroid precursors (E-cadherin+) in all 6 cases. One case also presented rare megakaryocytes, and one showed benign TdT+ B-cell precursors. To our knowledge, this is the first study that demonstrates EMH as a common finding in orchiectomy samples, especially from newborns. Despite the lack of pathologic potential, it is important to recognize EMH in order to avoid misdiagnosis.