Stanley L. Schrier

Human bone marrow hematopoietic stem cells are increased in frequency and myeloid-biased with age

In the human hematopoietic system, aging is associated with decreased bone marrow cellularity, decreased adaptive immune system function, and increased incidence of anemia and other hematological disorders and malignancies. Recent studies in mice suggest that changes within the hematopoietic stem cell (HSC) population during aging contribute significantly to the manifestation of these age-associated hematopoietic pathologies. Though the mouse HSC population has been shown to change both quantitatively and functionally with age, changes in the human HSC and progenitor cell populations during aging have been incompletely characterized. To elucidate the properties of an aged human hematopoietic system that may predispose to age-associated hematopoietic dysfunction, we evaluated immunophenotypic HSC and other hematopoietic progenitor populations from healthy, hematologically normal young and elderly human bone marrow samples. We found that aged immunophenotypic human HSC increase in frequency, are less quiescent, and exhibit myeloid-biased differentiation potential compared with young HSC. Gene expression profiling revealed that aged immunophenotypic human HSC transcriptionally up-regulate genes associated with cell cycle, myeloid lineage specification, and myeloid malignancies. These age-associated alterations in the frequency, developmental potential, and gene expression profile of human HSC are similar to those changes observed in mouse HSC, suggesting that hematopoietic aging is an evolutionarily conserved process.

Granulopoiesis in Acute Myeloid Leukemia and Preleukemia

Abstract In vitro regulation of granulocytic proliferation and differentiation was analyzed by study of marrow granulocytic colony-forming capacity (CFC) and peripheral white-cell provision of colony-stimulating activity (CSA) in patients with acute myeloid leukemia (AML) and preleukemia. Patients with AML in relapse had abnormal marrow CFC, producing either no or excessive numbers of abortive colonies. Furthermore, their leukocytes lacked ability to provide CSA. During complete remission, both these indexes were normal. Sequential studies indicated that marrow CFC correlates with, and probably precedes, detectable changes in clinical and morphologic status. Preleukemic patients showed disordered CFC and CSA, with values similar to those in AML in relapse. AML marrow appears to consist of coexisting normal and leukemic cell clones, and preleukemic marrow to contain either a potentially leukemic clone with greater capacity for differentiation in vivo or a leukemic clone that is held in abeyance.

Pathophysiology of thalassemia

Despite discoveries concerning the molecular abnormalities that led to the thalassemic syndromes, it still is not known how accumulation of excess unmatched &agr;-globin in &bgr; thalassemia and &bgr;-globin in &agr; thalassemia leads to red blood cell hemolysis in the peripheral blood, and in the &bgr; thalassemias particularly, premature destruction of erythroid precursors in marrow (ineffective erythropoiesis). Oxidant injury may cause hemolysis, but there is no evidence that it causes ineffective erythropoiesis. Hemoglobin E/&bgr; thalassemia is now a worldwide clinical problem. The reasons underlying the heterogeneity and occasional severity of the syndrome remain obscure. Ineffective erythropoiesis now appears to be caused by accelerated apoptosis, in turn caused primarily by deposition of &agr;-globin chains in erythroid precursors. However, it is not clear how &agr;-globin deposition causes apoptosis. The author uses new observations on the control of erythropoiesis to provide a framework for studying the enhanced thalassemic erythroid apoptosis.

Multivariate analysis of factors associated with invasive fungal disease during remission induction therapy for acute myelogenous leukemia

The clinical courses of 54 consecutive adult patients with acute myelogenous leukemia (AML) who underwent 67 courses of intensive remission induction therapy were analyzed to assess factors associated with development of serious fungal and bacterial infections. Fever developed in 65 of 67 remission induction attempts and was due to bacterial, bacterial‐fungal, and fungal etiologies in 49%, 14%, and 9% of cases, respectively. No etiology of fever was found in 28% of cases. Bacteremia occurred in 54% of remission induction attempts. Invasive fungal disease (IFD) occurred in 22% of cases with an overall mortality of 60%, including 45% of the patients who died during treatment. Using multivariate logistic regression analysis, a mathematical model was constructed which correlated with the risk of IFD. Major factors associated with patients who ultimately develop IFD included the duration of chemotherapy, the number of sites colonized with fungi and the number of fungal species isolated on certain surveillance cultures, particularly Aspergillus species. These studies define characteristics of patients at high risk for development of IFD for whom early initiation of empiric antifungal therapy is strongly recommended.

VEGF modulates erythropoiesis through regulation of adult hepatic erythropoietin synthesis

Vascular endothelial growth factor (VEGF) exerts crucial functions during pathological angiogenesis and normal physiology. We observed increased hematocrit (60–75%) after high-grade inhibition of VEGF by diverse methods, including adenoviral expression of soluble VEGF receptor (VEGFR) ectodomains, recombinant VEGF Trap protein and the VEGFR2-selective antibody DC101. Increased production of red blood cells (erythrocytosis) occurred in both mouse and primate models, and was associated with near-complete neutralization of VEGF corneal micropocket angiogenesis. High-grade inhibition of VEGF induced hepatic synthesis of erythropoietin (Epo, encoded by Epo) >40-fold through a HIF-1α–independent mechanism, in parallel with suppression of renal Epo mRNA. Studies using hepatocyte-specific deletion of the Vegfa gene and hepatocyte–endothelial cell cocultures indicated that blockade of VEGF induced hepatic Epo by interfering with homeostatic VEGFR2-dependent paracrine signaling involving interactions between hepatocytes and endothelial cells. These data indicate that VEGF is a previously unsuspected negative regulator of hepatic Epo synthesis and erythropoiesis and suggest that levels of Epo and erythrocytosis could represent noninvasive surrogate markers for stringent blockade of VEGF in vivo.NOTE: In the version of this article initially published, the name of one of the authors, Nihar R. Nayak, was misspelled as Nihar R. Niyak. The error has been corrected in the HTML and PDF versions of the article.

Multiple clinical forms of dehydrated hereditary stomatocytosis arise from mutations in PIEZO1

Autosomal dominant dehydrated hereditary stomatocytosis (DHSt) usually presents as a compensated hemolytic anemia with macrocytosis and abnormally shaped red blood cells (RBCs). DHSt is part of a pleiotropic syndrome that may also exhibit pseudohyperkalemia and perinatal edema. We identified PIEZO1 as the disease gene for pleiotropic DHSt in a large kindred by exome sequencing analysis within the previously mapped 16q23-q24 interval. In 26 affected individuals among 7 multigenerational DHSt families with the pleiotropic syndrome, 11 heterozygous PIEZO1 missense mutations cosegregated with disease. PIEZO1 is expressed in the plasma membranes of RBCs and its messenger RNA, and protein levels increase during in vitro erythroid differentiation of CD34(+) cells. PIEZO1 is also expressed in liver and bone marrow during human and mouse development. We suggest for the first time a correlation between a PIEZO1 mutation and perinatal edema. DHSt patient red cells with the R2456H mutation exhibit increased ion-channel activity. Functional studies of PIEZO1 mutant R2488Q expressed in Xenopus oocytes demonstrated changes in ion-channel activity consistent with the altered cation content of DHSt patient red cells. Our findings provide direct evidence that R2456H and R2488Q mutations in PIEZO1 alter mechanosensitive channel regulation, leading to increased cation transport in erythroid cells.

Hematopoietic stem cell and progenitor cell mechanisms in myelodysplastic syndromes.

Myelodysplastic syndromes (MDS) are a group of disorders characterized by variable cytopenias and ineffective hematopoiesis. Hematopoietic stem cells (HSCs) and myeloid progenitors in MDS have not been extensively characterized. We transplanted purified human HSCs from MDS samples into immunodeficient mice and show that HSCs are the disease-initiating cells in MDS. We identify a recurrent loss of granulocyte-macrophage progenitors (GMPs) in the bone marrow of low risk MDS patients that can distinguish low risk MDS from clinical mimics, thus providing a simple diagnostic tool. The loss of GMPs is likely due to increased apoptosis and increased phagocytosis, the latter due to the up-regulation of cell surface calreticulin, a prophagocytic marker. Blocking calreticulin on low risk MDS myeloid progenitors rescues them from phagocytosis in vitro. However, in the high-risk refractory anemia with excess blasts (RAEB) stages of MDS, the GMP population is increased in frequency compared with normal, and myeloid progenitors evade phagocytosis due to up-regulation of CD47, an antiphagocytic marker. Blocking CD47 leads to the selective phagocytosis of this population. We propose that MDS HSCs compete with normal HSCs in the patients by increasing their frequency at the expense of normal hematopoiesis, that the loss of MDS myeloid progenitors by programmed cell death and programmed cell removal are, in part, responsible for the cytopenias, and that up-regulation of the “don’t eat me” signal CD47 on MDS myeloid progenitors is an important transition step leading from low risk MDS to high risk MDS and, possibly, to acute myeloid leukemia.

Drug-induced erythrocyte membrane internalization

In vitro erythrocyte membrane internalization, resulting in the formation of membrane-lined vacuoles, can be quantified by a radioisotopic method. A complex of (37)Co-labeled vitamin B(12) and its plasma protein binders is first adsorbed to the cell surface, and after vacuoles are formed, the noninternalized label is removed by washing and trypsin treatment. The residual radioactivity represents trapped label and can be used to measure the extent of membrane internalization. Using this method, it was found that in addition to primaquine, a group of membrane-active drugs, specifically hydrocortisone, vinblastine, and chlorpromazine can induce membrane internalization in erythrocytes. This is a metabolic process dependent on drug concentration, temperature, and pH. Vacuole formation by all agents tested can be blocked by prior depletion of endogenous substrates or by poisoning the erythrocytes with sodium fluoride and sulfhydryl blocking agents. This phenomenon resembles in some respects the previously reported membrane internalization of energized erythrocyte ghosts. It is suggested that membrane internalization is dependent on an ATP-energized state and is influenced by the balance between the concentrations of magnesium and calcium in the membrane. This study provides a basis for proposing a unifying concept of the action of some membrane-active drugs, and for considering the role of erythrocyte membrane internalization in pathophysiologic events.

Evaluation and management of anemia in the elderly.

Anemia is now recognized as a risk factor for a number of adverse outcomes in the elderly, including hospitalization, morbidity, and mortality. What constitutes appropriate evaluation and management for an elderly patient with anemia, and when to initiate a referral to a hematologist, are significant issues. Attempts to identify suggested hemoglobin levels for blood transfusion therapy have been confounded for elderly patients with their co‐morbidities. Since no specific recommended hemoglobin threshold has stood the test of time, prudent transfusion practices to maintain hemoglobin thresholds of 9–10 g/dL in the elderly are indicated, unless or until evidence emerges to indicate otherwise. Am. J. Hematol. 89:88–96, 2014.

Acute myelogenous leukemia in two patients treated with azathioprine for nonmalignant diseases

Abstract Two patients receiving long-term azathioprine therapy for immunosuppression of nonmalignant conditions (chronic active hepatitis and renal transplantation) were admitted to Stanford University Hospital with acute myelogenous leukemia. Possible mechanisms of leukemogenesis are discussed. A review of the literature reveals these to be the first reported instances of acute myelogenous leukemia occurring after azathioprine immunosuppression for nonmalignant illness.