B-cell acute lymphoblastic leukemia with iAMP21 in a patient with Down syndrome due to a constitutional isodicentric chromosome 21.

Pediatric B-cell acute lymphoblastic leukemia (B-ALL) is associated with various specific cytogenetic and molecular markers that have significant influence on treatment and prognosis. A subset of children has a much higher risk of developing B-ALL due to constitutional genetic alterations such as trisomy 21 (Down's syndrome). In these patients, B-ALL is often associated with specific genomic profiles leading to leukemic transformation. In rare cases, constitutional structural chromosomal abnormalities involving chromosome 21, such as the der(15;21) Robertsonian translocation and a ring 21 chromosome, have been associated with intrachromosomal amplification of chromosome 21 (iAMP21) B-ALL. Here, we report the development of B-ALL in a child with Down's syndrome who carries a constitutional isodicentric chromosome 21 [idic(21)], described previously by Putra et al., 2017. This idic(21) appeared to be unstable during mitosis, leading to somatic rearrangements consistent with iAMP21 amplification, resulting in the development of leukemia. In this case, a single constitutional structural chromosome 21 rearrangement resulted in a B-ALL with Down syndrome-associated genomic lesions as well as genomic lesions not common to the Down syndrome subtype of B-ALL. Our findings highlight the need for counseling of individuals with constitutional structural chromosome 21 rearrangements regarding their risks of developing a B-ALL.

Interleukin-6 mediates neutrophil mobilization from bone marrow in pulmonary hypertension.

Myeloid cells, such as neutrophils, are produced in the bone marrow in high quantities and are important in the pathogenesis of vascular diseases such as pulmonary hypertension (PH). Although neutrophil recruitment into sites of inflammation has been well studied, the mechanisms of neutrophil egress from the bone marrow are not well understood. Using computational flow cytometry, we observed increased neutrophils in the lungs of patients and mice with PH. Moreover, we found elevated levels of IL-6 in the blood and lungs of patients and mice with PH. We observed that transgenic mice overexpressing Il-6 in the lungs displayed elevated neutrophil egress from the bone marrow and exaggerated neutrophil recruitment to the lungs, resulting in exacerbated pulmonary vascular remodeling, and dysfunctional hemodynamics. Mechanistically, we found that IL-6-induced neutrophil egress from the bone marrow was dependent on interferon regulatory factor 4 (IRF-4)-mediated CX3CR1 expression in neutrophils. Consequently, Cx3cr1 genetic deficiency in hematopoietic cells in Il-6-transgenic mice significantly reduced neutrophil egress from bone marrow and decreased neutrophil counts in the lungs, thus ameliorating pulmonary remodeling and hemodynamics. In summary, these findings define a novel mechanism of IL-6-induced neutrophil egress from the bone marrow and reveal a new therapeutic target to curtail neutrophil-mediated inflammation in pulmonary vascular disease.

Apoptosis of hematopoietic progenitor-derived adipose tissue-resident macrophages contributes to insulin resistance after myocardial infarction.

Patients with insulin resistance have high risk of cardiovascular disease such as myocardial infarction (MI). However, it is not known whether MI can initiate or aggravate insulin resistance. We observed that patients with ST-elevation MI and mice with MI had de novo hyperglycemia and features of insulin resistance, respectively. In mouse models of both myocardial and skeletal muscle injury, we observed that the number of visceral adipose tissue (VAT)-resident macrophages decreased because of apoptosis after these distant organ injuries. Patients displayed a similar decrease in VAT-resident macrophage numbers and developed systemic insulin resistance after ST-elevation MI. Loss of VAT-resident macrophages after MI injury led to systemic insulin resistance in non-diabetic mice. Danger signaling-associated protein high mobility group box 1 was released by the dead myocardium after MI in rodents and triggered macrophage apoptosis via Toll-like receptor 4. The VAT-resident macrophage population in the steady state in mice was transcriptomically distinct from macrophages in the brain, skin, kidney, bone marrow, lungs, and liver and was derived from hematopoietic progenitor cells just after birth. Mechanistically, VAT-resident macrophage apoptosis and de novo insulin resistance in mouse models of MI were linked to diminished concentrations of macrophage colony-stimulating factor and adiponectin. Collectively, these findings demonstrate a previously unappreciated role of adipose tissue-resident macrophages in sensing remote organ injury and promoting MI pathogenesis.

Nrf2 deficiency in mice attenuates erythropoietic stress-related macrophage hypercellularity.

Erythropoiesis in the bone marrow and spleen depends on intricate interactions between the resident macrophages and erythroblasts. Our study focuses on identifying the role of nuclear factor erythroid 2-related factor 2 (Nrf2) during recovery from stress erythropoiesis. To that end, we induced stress erythropoiesis in Nrf2+/+ and Nrf2-null mice and evaluated macrophage subsets known to support erythropoiesis and erythroid cell populations. Our results confirm macrophage and erythroid hypercellularity after acute blood loss. Importantly, Nrf2 depletion results in a marked numerical reduction of F4/80+/CD169+/CD11b+ macrophages, which is more prominent under the induction of stress erythropoiesis. The observed macrophage deficiency is concomitant to a significantly impaired erythroid response to acute stress erythropoiesis in both murine bone marrow and murine spleen. Additionally, peripheral blood reticulocyte count as a response to acute blood loss is delayed in Nrf2-deficient mice compared with age-matched controls (11.0 ± 0.6% vs. 14.8 ± 0.6%, p ≤ 0.001). Interestingly, we observe macrophage hypercellularity in conjunction with erythroid hyperplasia in the bone marrow during stress erythropoiesis in Nrf2+/+ controls, with both impaired in Nrf2-/- mice. We further confirm the finding of macrophage hypercellularity in another model of erythroid hyperplasia, the transgenic sickle cell mouse, characterized by hemolytic anemia and chronic stress erythropoiesis. Our results revealed the role of Nrf2 in stress erythropoiesis in the bone marrow and that macrophage hypercellularity occurs concurrently with erythroid expansion during stress erythropoiesis. Macrophage hypercellularity is a previously underappreciated feature of stress erythropoiesis in sickle cell disease and recovery from blood loss.

Cardiac expression of HMOX1 and PGF in sickle cell mice and haem-treated wild type mice dominates organ expression profiles via Nrf2 (Nfe2l2).

Haemolysis is a major feature of sickle cell disease (SCD) that contributes to organ damage. It is well established that haem, a product of haemolysis, induces expression of the enzyme that degrades it, haem oxygenase-1 (HMOX1). We have also shown that haem induces expression of placental growth factor (PGF), but the organ specificity of these responses has not been well-defined. As expected, we found high level expression of Hmox1 and Pgf transcripts in the reticuloendothelial system organs of transgenic sickle cell mice, but surprisingly strong expression in the heart (P < 0·0001). This pattern was largely replicated in wild type mice by intravenous injection of exogenous haem. In the heart, haem induced unexpectedly strong mRNA responses for Hmox1 (18-fold), Pgf (4-fold), and the haem transporter Slc48a1 (also termed Hrg1; 2·4-fold). This was comparable to the liver, the principal known haem-detoxifying organ. The NFE2L2 (also termed NRF2) transcription factor mediated much of the haem induction of Hmox1 and Hrg1 in all organs, but less so for Pgf. Our results indicate that the heart expresses haem response pathway genes at surprisingly high basal levels and shares with the liver a similar transcriptional response to circulating haem. The role of the heart in haem response should be investigated further.

Diagnostic Utility of Isolated Tube C Positivity in T-Cell Receptor ß Testing Using BIOMED-2 Primers.

OBJECTIVES: T-cell receptor (TCR) gene rearrangement studies are widely used for assessing T-cell clonality. The frequency and significance of clonal peaks restricted to TCR ß (TCRB) tube C are uncertain. We retrospectively reviewed 80 TCR studies performed on bone marrow/peripheral blood. METHODS: TCRB and TCR γ (TCRG) analyses were performed using BIOMED-2 primers. A peak was considered clonal or atypical if it was reproducible and 5× or more or 3× to 5× polyclonal background, respectively. RESULTS: TCRB analysis demonstrated 12 (15%) of 80 cases with one to four isolated peaks in tube C (>3×) with polyclonal pattern in tubes A and B. TCRG analysis was monoclonal in two cases (both definite T-cell neoplasms), polyclonal in four, and oligoclonal in six. Of the 10 cases without clone in TCRG, six had autoimmune disorder and none had T-cell neoplasm. CONCLUSIONS: Peaks restricted to TCRB tube C in the TCR analysis may be misleading, as it is often not indicative of an overt T-cell neoplasm.

Iron modulation of erythropoiesis is associated with Scribble-mediated control of the erythropoietin receptor.

Iron-restricted human anemias are associated with the acquisition of marrow resistance to the hematopoietic cytokine erythropoietin (Epo). Regulation of Epo responsiveness by iron availability serves as the basis for intravenous iron therapy in anemias of chronic disease. Epo engagement of its receptor normally promotes survival, proliferation, and differentiation of erythroid progenitors. However, Epo resistance caused by iron restriction selectively impairs proliferation and differentiation while preserving viability. Our results reveal that iron restriction limits surface display of Epo receptor in primary progenitors and that mice with enforced surface retention of the receptor fail to develop anemia with iron deprivation. A mechanistic pathway is identified in which erythroid iron restriction down-regulates a receptor control element, Scribble, through the mediation of the iron-sensing transferrin receptor 2. Scribble deficiency reduces surface expression of Epo receptor but selectively retains survival signaling via Akt. This mechanism integrates nutrient sensing with receptor function to permit modulation of progenitor expansion without compromising survival.

Nitric Oxide-Independent Soluble Guanylate Cyclase Activation Improves Vascular Function and Cardiac Remodeling in Sickle Cell Disease.

Sickle cell disease (SCD) is associated with intravascular hemolysis and oxidative inhibition of nitric oxide (NO) signaling. BAY 54-6544 is a small-molecule activator of oxidized soluble guanylate cyclase (sGC), which, unlike endogenous NO and the sGC stimulator, BAY 41-8543, preferentially binds and activates heme-free, NO-insensitive sGC to restore enzymatic cGMP production. We tested orally delivered sGC activator, BAY 54-6544 (17 mg/kg/d), sGC stimulator, BAY 41-8543, sildenafil, and placebo for 4-12 weeks in the Berkeley transgenic mouse model of SCD (BERK-SCD) and their hemizygous (Hemi) littermate controls (BERK-Hemi). Right ventricular (RV) maximum systolic pressure (RVmaxSP) was measured using micro right-heart catheterization. RV hypertrophy (RVH) was determined using Fulton's index and RV corrected weight (ratio of RV to tibia). Pulmonary artery vasoreactivity was tested for endothelium-dependent and -independent vessel relaxation. Right-heart catheterization revealed higher RVmaxSP and RVH in BERK-SCD versus BERK-Hemi, which worsened with age. Treatment with the sGC activator more effectively lowered RVmaxSP and RVH, with 90-day treatment delivering superior results, when compared with other treatments and placebo groups. In myography experiments, acetylcholine-induced (endothelium-dependent) and sodium-nitroprusside-induced (endothelium-independent NO donor) relaxation of the pulmonary artery harvested from placebo-treated BERK-SCD was impaired relative to BERK-Hemi but improved after therapy with sGC activator. By contrast, no significant effect for sGC stimulator or sildenafil was observed in BERK-SCD. These findings suggest that sGC is oxidized in the pulmonary arteries of transgenic SCD mice, leading to blunted responses to NO, and that the sGC activator, BAY 54-6544, may represent a novel therapy for SCD-associated pulmonary arterial hypertension and cardiac remodeling.

Induction of deubiquitinating enzyme USP50 during erythropoiesis and its potential role in the regulation of Ku70 stability.

Anemia is a very common blood disorder that affects the lives of billions of people worldwide. Anemia is caused by the loss of blood, increased destruction of red blood cells (RBCs), or reduced production of RBCs. Erythropoiesis is the complex process of RBC differentiation and maturation, in which protein degradation plays a crucial role. Protein ubiquitination regulates programmed protein degradation, which can be reversed by deubiquitinating enzymes (DUBs); however, the role of DUBs in erythropoiesis has not been well studied. We examined the expression of DUBs during erythropoiesis using an ex vivo human CD34+ hematopoietic progenitor cell culture system. Here we show that ubiquitin-specific protease 50 (USP50) levels are increased during erythropoiesis. USP50 mRNA levels are significantly increased on day 3 and protein levels are elevated on day 9 of erythroid differentiation. Coimmunoprecipitation and proteomics analyses reveal that Ku70, a DNA-binding protein, is associated with USP50. Overexpression of USP50 has no effect on Ku70 mRNA levels, while it reduces Ku70 protein levels by promoting Ku70 degradation, suggesting that USP50 may indirectly regulate Ku70 protein stability. USP50 protein is also not stable. USP50 protein degradation is independent of the proteasomal and the lysosomal degradation systems. This study suggests that DUBs like USP50 may regulate protein stability during erythropoiesis; however, more investigation is warranted.

Multiple cutaneous lymphoproliferative disorders showing a retained tumor clone by T-cell receptor gene rearrangement analysis: a case series of four patients and review of the literature.

BACKGROUND: Mycosis fungoides (MF) is the most common primary cutaneous T-cell lymphoma (CTCL), followed by CD30+ lymphoproliferative disorders, including lymphomatoid papulosis (LyP) and primary cutaneous anaplastic large cell lymphoma (pcALCL). The objective was to report on a series of patients with different types of CTCL at different times in their clinical course, with a focus on clonality studies. METHODS: Four patients with multiple diagnoses of CTCLs were identified. The clinical information, treatment interventions, and histopathology were reviewed. T-cell receptor (TCR) gene rearrangement studies were performed on all available specimens. RESULTS: The four patients carried diagnoses of: (1) pcALCL and MF; (2) pcALCL, LyP, and pcALCL; (3) LyP, MF, and pcALCL; (4) LyP, pcALCL, and MF; each with characteristic presentation and histopathologic findings. The results of the TCR polymerase chain reaction showed that all tumors expressed and retained a TCR clone(s) as follows: (1) biallelic clone; (2) single clone; (3) biallelic clone with additional clone; and (4) single clone, respectively. CONCLUSION: We report a series of four cases of individual patients with coexisting diagnoses of some combination of MF, LyP, and pcALCL, whose lesions presented in nontraditional sequence and demonstrated a retained clone by gene rearrangement analysis.

Platelet-derived HMGB1 is a critical mediator of thrombosis.

Thrombosis and inflammation are intricately linked in several major clinical disorders, including disseminated intravascular coagulation and acute ischemic events. The damage-associated molecular pattern molecule high-mobility group box 1 (HMGB1) is upregulated by activated platelets in multiple inflammatory diseases; however, the contribution of platelet-derived HMGB1 in thrombosis remains unexplored. Here, we generated transgenic mice with platelet-specific ablation of HMGB1 and determined that platelet-derived HMGB1 is a critical mediator of thrombosis. Mice lacking HMGB1 in platelets exhibited increased bleeding times as well as reduced thrombus formation, platelet aggregation, inflammation, and organ damage during experimental trauma/hemorrhagic shock. Platelets were the major source of HMGB1 within thrombi. In trauma patients, HMGB1 expression on the surface of circulating platelets was markedly upregulated. Moreover, evaluation of isolated platelets revealed that HMGB1 is critical for regulating platelet activation, granule secretion, adhesion, and spreading. These effects were mediated via TLR4- and MyD88-dependent recruitment of platelet guanylyl cyclase (GC) toward the plasma membrane, followed by MyD88/GC complex formation and activation of the cGMP-dependent protein kinase I (cGKI). Thus, we establish platelet-derived HMGB1 as an important mediator of thrombosis and identify a HMGB1-driven link between MyD88 and GC/cGKI in platelets. Additionally, these findings suggest a potential therapeutic target for patients sustaining trauma and other inflammatory disorders associated with abnormal coagulation.

Red Blood Cells Store and Release Interleukin-33.

Interleukin-33 (IL-33) is a member of the IL-1 cytokine superfamily that potently drives production of a variety of cytokines and contributes to the pathogenesis of inflammatory diseases. The IL-33 is a nuclear protein and is released from apoptotic or necrotic cells. Serum IL-33 levels are increased in various diseases, such as atopic dermatitis, chronic hepatitis C infection, and asthma. Here, we show that red blood cells (RBCs) are one of the major sources of plasma IL-33. The IL-33 levels are significantly increased in supernatants from lysed RBCs. Plasma IL-33 levels are increased in patients during hemolysis, and plasma IL-33 levels show a positive correlation with degree of hemolysis. The IL-33 protein and messenger RNA levels were detected in the late stages of differentiation in ex vivo primary human erythroid progenitor cell cultures, suggesting that IL-33 is expressed during maturation of RBCs. Furthermore, hemoglobin depleted red cell lysates induced IL-8 expression in human epithelial cells. This effect was attenuated in IL-33 decoy receptor expressing cells and was enhanced in IL-33 receptor expressing cells. These results suggest that erythroid progenitor cells produce IL-33 and circulating RBCs represent a major source of IL-33 that is released upon hemolysis.

Isocitrate ameliorates anemia by suppressing the erythroid iron restriction response.

The unique sensitivity of early red cell progenitors to iron deprivation, known as the erythroid iron restriction response, serves as a basis for human anemias globally. This response impairs erythropoietin-driven erythropoiesis and underlies erythropoietic repression in iron deficiency anemia. Mechanistically, the erythroid iron restriction response results from inactivation of aconitase enzymes and can be suppressed by providing the aconitase product isocitrate. Recent studies have implicated the erythroid iron restriction response in anemia of chronic disease and inflammation (ACDI), offering new therapeutic avenues for a major clinical problem; however, inflammatory signals may also directly repress erythropoiesis in ACDI. Here, we show that suppression of the erythroid iron restriction response by isocitrate administration corrected anemia and erythropoietic defects in rats with ACDI. In vitro studies demonstrated that erythroid repression by inflammatory signaling is potently modulated by the erythroid iron restriction response in a kinase-dependent pathway involving induction of the erythroid-inhibitory transcription factor PU.1. These results reveal the integration of iron and inflammatory inputs in a therapeutically tractable erythropoietic regulatory circuit.

Protein kinase D-HDAC5 signaling regulates erythropoiesis and contributes to erythropoietin cross-talk with GATA1.

In red cell development, the differentiation program directed by the transcriptional regulator GATA1 requires signaling by the cytokine erythropoietin, but the mechanistic basis for this signaling requirement has remained unknown. Here we show that erythropoietin regulates GATA1 through protein kinase D activation, promoting histone deacetylase 5 (HDAC5) dissociation from GATA1, and subsequent GATA1 acetylation. Mice deficient for HDAC5 show resistance to anemic challenge and altered marrow responsiveness to erythropoietin injections. In ex vivo studies, HDAC5(-/-) progenitors display enhanced entry into and passage through the erythroid lineage, as well as evidence of erythropoietin-independent differentiation. These results reveal a molecular pathway that contributes to cytokine regulation of hematopoietic differentiation and offer a potential mechanism for fine tuning of lineage-restricted transcription factors by lineage-specific cytokines.

Aconitase regulation of erythropoiesis correlates with a novel licensing function in erythropoietin-induced ERK signaling.

BACKGROUND: Erythroid development requires the action of erythropoietin (EPO) on committed progenitors to match red cell output to demand. In this process, iron acts as a critical cofactor, with iron deficiency blunting EPO-responsiveness of erythroid progenitors. Aconitase enzymes have recently been identified as possible signal integration elements that couple erythropoiesis with iron availability. In the current study, a regulatory role for aconitase during erythropoiesis was ascertained using a direct inhibitory strategy. METHODOLOGY/PRINCIPAL FINDINGS: In C57BL/6 mice, infusion of an aconitase active-site inhibitor caused a hypoplastic anemia and suppressed responsiveness to hemolytic challenge. In a murine model of polycythemia vera, aconitase inhibition rapidly normalized red cell counts, but did not perturb other lineages. In primary erythroid progenitor cultures, aconitase inhibition impaired proliferation and maturation but had no effect on viability or ATP levels. This inhibition correlated with a blockade in EPO signal transmission specifically via ERK, with preservation of JAK2-STAT5 and Akt activation. Correspondingly, a physical interaction between ERK and mitochondrial aconitase was identified and found to be sensitive to aconitase inhibition. CONCLUSIONS/SIGNIFICANCE: Direct aconitase inhibition interferes with erythropoiesis in vivo and in vitro, confirming a lineage-selective regulatory role involving its enzymatic activity. This inhibition spares metabolic function but impedes EPO-induced ERK signaling and disturbs a newly identified ERK-aconitase physical interaction. We propose a model in which aconitase functions as a licensing factor in ERK-dependent proliferation and differentiation, thereby providing a regulatory input for iron in EPO-dependent erythropoiesis. Directly targeting aconitase may provide an alternative to phlebotomy in the treatment of polycythemia vera.

Posttransplantation lymphoproliferative disease involving the pituitary gland.

Posttransplantation lymphoproliferative disorders (PTLD) are heterogeneous lesions with variable morphology, immunophenotype, and molecular characteristics. Multiple distinct primary lesions can occur in PTLD, rarely with both B-cell and T-cell characteristics. Lesions can involve both grafted organs and other sites; however, PTLD involving the pituitary gland has not been previously reported. We describe a patient who developed Epstein-Barr virus-negative PTLD 13 years posttransplantation involving the terminal ileum and pituitary, which was simultaneously involved by a pituitary adenoma. Immunohistochemistry of the pituitary lesion showed expression of CD79a, CD3, and CD7 with clonal rearrangements of both T-cell receptor gamma chain (TRG@) and immunoglobulin heavy chain (IGH@) genes. The terminal ileal lesion was immunophenotypically and molecularly distinct. This is the first report of pituitary PTLD and illustrates the potentially complex nature of PTLD.

Iron control of erythroid development by a novel aconitase-associated regulatory pathway.

Human red cell differentiation requires the action of erythropoietin on committed progenitor cells. In iron deficiency, committed erythroid progenitors lose responsiveness to erythropoietin, resulting in hypoplastic anemia. To address the basis for iron regulation of erythropoiesis, we established primary hematopoietic cultures with transferrin saturation levels that restricted erythropoiesis but permitted granulopoiesis and megakaryopoiesis. Experiments in this system identified as a critical regulatory element the aconitases, multifunctional iron-sulfur cluster proteins that metabolize citrate to isocitrate. Iron restriction suppressed mitochondrial and cytosolic aconitase activity in erythroid but not granulocytic or megakaryocytic progenitors. An active site aconitase inhibitor, fluorocitrate, blocked erythroid differentiation in a manner similar to iron deprivation. Exogenous isocitrate abrogated the erythroid iron restriction response in vitro and reversed anemia progression in iron-deprived mice. The mechanism for aconitase regulation of erythropoiesis most probably involves both production of metabolic intermediates and modulation of erythropoietin signaling. One relevant signaling pathway appeared to involve protein kinase Calpha/beta, or possibly protein kinase Cdelta, whose activities were regulated by iron, isocitrate, and erythropoietin.

Cross-talk of GATA-1 and P-TEFb in megakaryocyte differentiation.

The transcription factor GATA-1 participates in programming the differentiation of multiple hematopoietic lineages. In megakaryopoiesis, loss of GATA-1 function produces complex developmental abnormalities and underlies the pathogenesis of megakaryocytic leukemia in Down syndrome. Its distinct functions in megakaryocyte and erythroid maturation remain incompletely understood. In this study, we identified functional and physical interaction of GATA-1 with components of the positive transcriptional elongation factor P-TEFb, a complex containing cyclin T1 and the cyclin-dependent kinase 9 (Cdk9). Megakaryocytic induction was associated with dynamic changes in endogenous P-TEFb composition, including recruitment of GATA-1 and dissociation of HEXIM1, a Cdk9 inhibitor. shRNA knockdowns and pharmacologic inhibition both confirmed contribution of Cdk9 activity to megakaryocytic differentiation. In mice with megakaryocytic GATA-1 deficiency, Cdk9 inhibition produced a fulminant but reversible megakaryoblastic disorder reminiscent of the transient myeloproliferative disorder of Down syndrome. P-TEFb has previously been implicated in promoting elongation of paused RNA polymerase II and in programming hypertrophic differentiation of cardiomyocytes. Our results offer evidence for P-TEFb cross-talk with GATA-1 in megakaryocytic differentiation, a program with parallels to cardiomyocyte hypertrophy.