Myeloid Precursor Cells Research Articles

Palmitoylation‐dependent endosomal localization of AATYK1A and its interaction with Src

Apoptosis-associated tyrosine kinase 1 (AATYK1), also named LMTK1, was previously isolated as an apoptosis-related gene from 32Dcl3 myeloid precursor cells, but its precise function remains unknown. AATYK1A, an isoform without a transmembrane domain, is highly expressed in neurons. We identified palmitoylation of AATYK1A at three N-terminal cysteine residues in cortical cultured neurons and COS-7 cells and found that palmitoylation determined localization of AATYK1A to the transferrin receptor-positive recycling endosomes. Further, we identified the tyrosine kinase Src as a novel AATYK1A-interacting protein. Src and Fyn phosphorylated AATYK1A at tyrosines 25 and 46 in a palmitoylation-dependent manner. The association of AATYK1A with Src in endosomes was also found to be palmitoylation-dependent. These results indicate that palmitoylation is a critical factor not only for the subcellular localization of AATYK1A but also for its interaction with Src.

Severe antibody‐mediated agranulocytosis

A 56-year-old female with Crohn's disease was admitted to the hospital with malaise, fever, and a low white blood cell count (0.8 x 10(9)/l) with no granulocytes or myeloid precursor cells in the bone marrow. The leucopenia was initially thought to be the result of an infection and she was treated with antibiotics and granulocyte colony-stimulating factor (G-CSF, filgrastim). However, the bacterial cultures and viral tests were all negative. The patient's condition deteriorated and she became morbidly ill, but recovered after high dose steroid treatment. Six weeks later she relapsed whilst receiving 7.5 mg daily dose of prednisolone. She recovered quickly after being given high dose methylprednisolone in combination with filgrastim. A high maintenance dose of prednisolone was tapered over 5 months. She has not relapsed since and is currently well. Antibodies to the human neutrophil antigen (HNA)-3a were detected, but these antibodies could not easily explain her agranulocytosis as she had a HNA-3a negative phenotype. It seems plausible that her agranulocytosis was immune mediated through autoantibodies directed towards the early myeloid cells.

Osteoclasts—the innate immune cells of the bone

Osteopenia and periarticular bony erosion are consequences of chronic inflammatory autoimmune disease due to an imbalance of osteoclast activity relative to new bone formation. Osteoclasts, which are specialized as the only bone resorbing cell type, are differentiated from hematopoietic myeloid precursor cells. Inflammatory signals mediated by multiple types of immune cells and cytokines have significant influence over osteoclast differentiation and function through direct effects on osteoclast precursors and indirect effects via osteoblasts and other cells in the bony microenvironment including synovial cells, stromal cells, osteocytes and chondrocytes. Recent studies have demonstrated that osteoclasts themselves express a number of immune receptors and are regulated similarly to macrophages and dendritic cells, closely related cells in the innate immune system. Though we are only beginning to understand the roles of innate immune receptors in osteoclasts, some of these receptors have been shown to be critical regulators of differentiation and function of osteoclasts. Osteoclasts likely function as the innate immune cells of the bone, thus are highly regulated to appropriately respond to stress and inflammatory changes in their microenvironment.

Mast cells are an essential hematopoietic component for polyp development

It is generally agreed that most colon cancers develop from adenomatous polyps, and it is this fact on which screening strategies are based. Although there is overwhelming evidence to link intrinsic genetic lesions with the formation of these preneoplastic lesions, recent data suggest that the tumor stromal environment also plays an essential role in this disease. In particular, it has been suggested that CD34(+) immature myeloid precursor cells are required for tumor development and invasion. Here we have used mice conditional for the stabilization of beta-catenin or defective for the adenomatous polyposis coli (APC) gene to reinvestigated the identity and importance of tumor-infiltrating hematopoietic cells in polyposis. We show that, from the onset, polyps are infiltrated with proinflammatory mast cells (MC) and their precursors. Depletion of MC either pharmacologically or through the generation of chimeric mice with genetic lesions in MC development leads to a profound remission of existing polyps. Our data suggest that MC are an essential hematopoietic component for preneoplastic polyp development and are a novel target for therapeutic intervention.

The divergent roles of alternatively activated macrophages in helminthic infections

Macrophages play crucial roles in the immune response, as they can initiate, modulate and also be final effector cells during immune responses to infections. Macrophages are derived from myeloid precursor cells in bone marrow and are widely distributed in every tissue of the body. Over the past 10 years, the concepts about macrophage activation have clearly changed; macrophages are not called activated or inactivated as they used to be. These changes in the concept of macrophage response is the result of many in vitro and in vivo studies, but the major support for the current concept of alternatively activated macrophages (AAMphi) comes from parasitic helminth infections. Parasitic helminths have developed complex mechanisms to evade and modulate host immunity. Infections with these parasites induce strong polarized Th2-type immune responses frequently associated with impaired T-cell proliferative responses to parasitic or unrelated antigens. Given the recent advances in understanding the immunoregulatory capabilities of helminthic infections, it has been suggested that macrophages can be a target for immunomodulation. Furthermore, they become altered when a host experiences chronic exposure to helminth parasites or their by-products, which favour the induction of AAMphi. How AAMphi participate in modulating host immunity during helminth infections and what their roles are in clearing or favouring parasite survival remains elusive. Here we review the most recent advances in the literature on AAMphi at the host-parasite interface, including three classes of helminths: nematodes (Brugia, Nippostrongylus, Litomosoides, Heligmosomoides), trematodes (Schistosoma, Fasciola) and cestodes (Taenia, Echinococcus, Hymenolepis).

Requirement of the E2F3 Transcription Factor for BCR/ABL Leukemogenesis.

Several RNA binding proteins (RBPs) have been implicated in the progression of chronic myelogenous leukemia (CML) from the indolent chronic phase to the aggressively fatal blast crisis. In the latter phase, expression and function of specific RBPs are altered at transcriptional or post-translational levels by the increased constitutive kinase activity of the BCR/ABL oncoprotein, resulting in enhanced resistance to apoptotic stimuli, growth advantage and differentiation arrest of CD34+ CML blast crisis (CML-BC) progenitors. In the current study, we identified by RIP (RNA immunoprecipitation)-mediated microarray analysis that mRNA encoding the E2F3 transcription factor associates to the BCR/ABL-regulated RBP hnRNP A1. Moreover, RNA electrophoretic mobility shift and UV-crosslinking assays revealed that hnRNP A1 interacts with E2F3 mRNA through a binding site located in the 3'UTR of both human and mouse E2F3 mRNA. Accordingly, E2F3 protein levels were upregulated in BCR/ABL-transformed myeloid precursor cell lines compared to parental cells in a BCR/ABL-kinase- and hnRNP A1 shuttling-dependent manner. In fact, treatment of BCR/ABL-expressing myeloid precursors with the kinase inhibitor Imatinib (2mM, 24 hr) or introduction of a dominant-negative shuttling-deficient hnRNP A1 protein (NLS-A1) markedly reduced E2F3 protein and mRNA levels. Similarly, upregulation of BCR/ABL expression/activity in the doxycycline inducible TonB2.10 cell line resulted in increased E2F3 protein expression. BCR/ABL kinase-dependent induction of E2F3 protein levels was also detected in CML-BCCD34+ compared to CML-CPCD34+ progenitors from paired patient samples and to normal CD34+ bone marrow samples. Importantly, the in vitro clonogenic potential of primary mouse BCR/ABL+ lineage negative (Lin−) progenitors was markedly impaired in BCR/ABL+ E2F3−/− compared to BCR/ABL-transduced E2F3+/+ myeloid progenitors and upon shRNA-mediated downregulation of E2F3 expression (90% inhibition, P<0.001). Furthermore, subcutaneous injection of shE2F3-expressing BCR/ABL+ cells into SCID mice markedly impaired in vivo tumorigenesis (>80% reduction in tumor burden, P<0.01). Accordingly, BCR/ABL leukemogenesis was strongly inhibited in SCID mice intravenously injected with E2F3 shRNA-expressing 32D-BCR/ABL cells and in mice transplanted with BCR/ABL-transduced Lin− bone marrow cells from E2F3−/− mice. Specifically, we demonstrate that reduced or absent levels of E2F3 resulted in dramatically decreased numbers of circulating BCR/ABL+ cells as determined by nested RT-PCR at 4 weeks post-injection (P=0.0001), normal splenic architecture and bone marrow cellularity and the absence of infiltrating myeloid blasts into non-hematopoietic compartments (i.e. liver). By contrast, SCID mice transplanted with vector-transduced 32D-BCR/ABL cells or BCR/ABL+ E2F3+/+ Lin− BM progenitors showed signs of an overt acute leukemia-like process with blast infiltration of hematopoietic and non-hematopoietic organs. Altogether, these data outline the importance of E2F3 expression for BCR/ABL leukemogenesis and characterize a new potential therapeutic target for the treatment of patients with advanced phase CML.

Presence of myeloid precursor cells in the endometrium of an AML patient: a diagnostic challenge!

Presence of myeloid precursor cells in the endometrium of an AML patient: a diagnostic challenge!

PML-Retinoic Acid Receptor α Inhibits PML IV Enhancement of PU.1-Induced C/EBPε Expression in Myeloid Differentiation

PML and PU.1 play important roles in myeloid differentiation. PML-deficient mice have an impaired capacity for terminal maturation of their myeloid precursor cells. This finding has been explained, at least in part, by the lack of PML action to modulate retinoic acid-differentiating activities. In this study, we found that C/EBPepsilon expression is reduced in PML-deficient mice. We showed that PU.1 directly activates the transcription of the C/EBPepsilon gene that is essential for granulocytic differentiation. The type IV isoform of PML interacted with PU.1, promoted its association with p300, and then enhanced PU.1-induced transcription and granulocytic differentiation. In contrast to PML IV, the leukemia-associated PML-retinoic acid receptor alpha fusion protein dissociated the PU.1/PML IV/p300 complex and inhibited PU.1-induced transcription. These results suggest a novel pathogenic mechanism of the PML-retinoic acid receptor alpha fusion protein in acute promyelocytic leukemia.

A Novel FLT3 Inhibitor FI-700 Selectively Suppresses the Growth of Leukemia Cells with FLT3 Mutations

The aim of this study was to evaluate the antileukemia activity of a novel FLT3 kinase inhibitor, FI-700. The antileukemia activity of FI-700 was evaluated in human leukemia cell lines, mutant or wild-type (Wt)-FLT3-expressing mouse myeloid precursor cell line, 32D and primary acute myeloid leukemia cells, and in xenograft or syngeneic mouse leukemia models. FI-700 showed a potent IC(50) value against FLT3 kinase at 20 nmol/L in an in vitro kinase assay. FI-700 showed selective growth inhibition against mutant FLT3-expressing leukemia cell lines and primary acute myeloid leukemia cells, whereas it did not affect the FLT3 ligand (FL)-driven growth of Wt-FLT3-expressing cells. These antileukemia activities were induced by the significant dephosphorylations of mutant FLT3 and STAT5, which resulted in G(1) arrest of the cell cycle. Oral administration of FI-700 induced the regression of tumors in a s.c. tumor xenograft model and increased the survival of mice in an i.v. transplanted model. Furthermore, FI-700 treatment eradicated FLT3/ITD-expressing leukemia cells, both in the peripheral blood and in the bone marrow. In this experiment, the depletion of FLT3/ITD-expressing cells by FI-700 was more significant than that of Ara-C, whereas bone marrow suppression by FI-700 was lower than that by Ara-C. FI-700 is a novel and potent FLT3 inhibitor with promising antileukemia activity.

Granulocytic sarcoma manifested as a parametrial mass mimicking a haemorrhagic abscess: a case report with CT and MR findings

Granulocytic sarcoma is a neoplasm arising from myeloid precursor cells and frequently accompanies leukaemia and myeloproliferative disorders. Granulocytic sarcoma can arise anywhere, and it frequently involves bones, perineural tissues and lymph nodes. However, granulocytic sarcoma in the female genital organs is uncommon, and it is extremely rare that it presents as an adnexal or parametrial mass. We report here the CT and MR findings in a case of granulocytic sarcoma that manifested as a uterine cervical and parametrial mass mimicking a haemorrhagic abscess in a 50-year-old woman with chronic myelogenous leukaemia.

The cell growth, morphology and immunocytochemistry of novel cell line established from a bone marrow of the patient with therapy-related myelodysplastic syndrome, entitled PC-MDS

We report on cell growth, morphology, and immunocytochemistry of the first human cell line, PC-MDS, derived from a bone marrow of a patient with therapy-related myelodysplastic syndrome who had no overt leukemia post-MDS phase. This cell population consisted of fast-growing mononuclear cells. Standard cytochemistry methods for detection of MPO, lipids, glycogen and ANAE gave results as follows: MPO and SBB negative while PAS and ANAE positive. Positive cytochemical staining and immunophenotype analyses indicated that PC-MDS cells have some characteristics of the early myeloid precursor cell. As the first t-MDS derived cell line it could be a new tool in evaluation of complex biology of MDS and also serves as a model for diverse in-vitro research.

PMA-Induced Differentiation of a Bone Marrow Progenitor Cell Line Activates HIV-1 LTR-Driven Transcription

Cells of the monocyte-macrophage lineage play an important role in human immunodeficiency virus type 1 (HIV-1)-associated disease. Infected myeloid precursor cells of the bone marrow are thought to be a viral reservoir that may repopulate the peripheral blood, central nervous system (CNS), and other organ systems throughout the course of disease. To model select aspects of HIV-1 infection of the bone marrow compartment in vitro, the erythro-myeloid precursor cell line, TF-1, was used. Phorbol 12-myristate 13-acetate (PMA) was found to induce the TF-1 cell line to differentiate through the myeloid lineage and become activated, as demonstrated by cellular morphologic changes and surface expression of differentiation and activation markers. Herein we demonstrate that HIV-1 long terminal repeats (LTRs) from T-, M-, and dual-tropic molecular clones have similar basal LTR activity in TF-1 cells and that differentiation of these cells by PMA resulted in increased LTR activity. Examination of specific cis-acting elements involved in basal and PMA-induced LTR activity demonstrated that the transcription factor families nuclear factor-kappa B (NF-kappaB) and specificity protein (Sp) contributed to the LTR activity of TF-1 cells, the Sp family being the most critical. These studies elucidate the impact of infected bone marrow monocytic cell differentiation on LTR activity and its potential impact on HIV-1-associated disease.

Tumor Exosomes Inhibit Differentiation of Bone Marrow Dendritic Cells

The production of exosomes by tumor cells has been implicated in tumor-associated immune suppression. In this study, we show that, in mice, exosomes produced by TS/A murine mammary tumor cells target CD11b(+) myeloid precursors in the bone marrow (BM) in vivo, and that this is associated with an accumulation of myeloid precursors in the spleen. Moreover, we demonstrate that TS/A exosomes block the differentiation of murine myeloid precursor cells into dendritic cells (DC) in vitro. Addition of tumor exosomes at day 0 led to a significant block of differentiation into DC, whereas addition at later time points was less effective. Similarly, exosomes produced by human breast tumor cells inhibited the differentiation of human monocytes in vitro. The levels of IL-6 and phosphorylated Stat3 were elevated 12 h after the tumor exosome stimulation of murine myeloid precursors, and tumor exosomes were less effective in inhibiting differentiation of BM cells isolated from IL-6 knockout mice. Addition of a rIL-6 to the IL-6 knockout BM cell culture restored the tumor exosome-mediated inhibition of DC differentiation. These data suggest that tumor exosome-mediated induction of IL-6 plays a role in blocking BM DC differentiation.

TREM2-Transduced Myeloid Precursors Mediate Nervous Tissue Debris Clearance and Facilitate Recovery in an Animal Model of Multiple Sclerosis

BackgroundIn multiple sclerosis, inflammation can successfully be prevented, while promoting repair is still a major challenge. Microglial cells, the resident phagocytes of the central nervous system (CNS), are hematopoietic-derived myeloid cells and express the triggering receptor expressed on myeloid cells 2 (TREM2), an innate immune receptor. Myeloid cells are an accessible source for ex vivo gene therapy. We investigated whether myeloid precursor cells genetically modified to express TREM2 affect the disease course of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis.Methods and FindingsEAE was induced in mice by immunization with a myelin autoantigen. Intravenous application of TREM2-transduced bone marrow–derived myeloid precursor cells at the EAE peak led to an amelioration of clinical symptoms, reduction in axonal damage, and prevention of further demyelination. TREM2-transduced myeloid cells applied intravenously migrated into the inflammatory spinal cord lesions of EAE-diseased mice, showed increased lysosomal and phagocytic activity, cleared degenerated myelin, and created an anti-inflammatory cytokine milieu within the CNS.ConclusionsIntravenously applied bone marrow–derived and TREM2-tranduced myeloid precursor cells limit tissue destruction and facilitate repair within the murine CNS by clearance of cellular debris during EAE. TREM2 is a new attractive target for promotion of repair and resolution of inflammation in multiple sclerosis and other neuroinflammatory diseases.

Rbm15 Modulates Notch-Induced Transcriptional Activation and Affects Myeloid Differentiation

RBM15 is the fusion partner with MKL in the t(1;22) translocation of acute megakaryoblastic leukemia. To understand the role of the RBM15-MKL1 fusion protein in leukemia, we must understand the normal functions of RBM15 and MKL. Here, we show a role for Rbm15 in myelopoiesis. Rbm15 is expressed at highest levels in hematopoietic stem cells and at more moderate levels during myelopoiesis of murine cell lines and primary murine cells. Decreasing Rbm15 levels with RNA interference enhances differentiation of the 32DWT18 myeloid precursor cell line. Conversely, enforced expression of Rbm15 inhibits 32DWT18 differentiation. We show that Rbm15 alters Notch-induced HES1 promoter activity in a cell type-specific manner. Rbm15 inhibits Notch-induced HES1 transcription in nonhematopoietic cells but stimulates this activity in hematopoietic cell lines, including 32DWT18 and human erythroleukemia cells. Moreover, the N terminus of Rbm15 coimmunoprecipitates with RBPJkappa, a critical factor in Notch signaling, and the Rbm15 N terminus has a dominant negative effect, impairing activation of HES1 promoter activity by full-length-Rbm15. Thus, Rbm15 is differentially expressed during hematopoiesis and may act to inhibit myeloid differentiation in hematopoietic cells via a mechanism that is mediated by stimulation of Notch signaling via RBPJkappa.

Tumor exosomes inhibit differentiation of bone marrow dendritic cells (49.14)

Abstract The production of exosomes by tumor cells has been implicated in tumor-associated immune suppression. Here we show that, in mice, exosomes produced by TS/A murine mammary tumor cells target CD11b+Gr-1+ myeloid precursors in the bone marrow in vivo and that this is associated with an accumulation of myeloid precursors in the spleen. Moreover, we demonstrate that TS/A exosomes block differentiation of murine myeloid precursor cells into dendritic cells in vitro. Addition of tumor exosomes at day 0 led to a complete block of differentiation into dendritic cells, whereas addition at later time points was less effective. Similarly, exosomes produced by human breast tumor cells inhibited differentiation of human monocytes in vitro. The levels of IL-6 and phosphorylated Stat3 were elevated 12 h after tumor exosome stimulation of murine myeloid precursors, and tumor exosomes were less effective in inhibiting differentiation of bone marrow cells isolated from IL-6 knockout mice. These data suggest that tumor exosome-mediated induction of IL-6 plays a role in blocking bone marrow dendritic cell differentiation.

Characteristics of novel myeloid precursor cell line, PC-MDS, established from a bone marrow of the patient with therapy-related myelodysplastic syndrome

We report on characteristics of the first human cell line, PC-MDS, derived from a bone marrow of a patient with therapy-related myelodysplastic syndrome (t-MDS) who had no overt post-MDS leukemia. Classic cytology analyses, immunophenotyping, cytogenetic and molecular genetic procedures were used for characterization of the cell line. PC-MDS cells are positive for the expression of CD13, CD15, CD30, CD33, and CD45 antigen. Positive cytochemical staining and immunophenotype analyses indicated that PC-MDS cells have some characteristics of the early myeloid precursor cell. The karyotype analysis of PC-MDS cell line revealed various numerical and structural changes including those typically associated with t-MDS: del(5)(q13)[7], der(5)t(5;11)(p11;q11)[13], −7[6], del(7)(q31)[2], +20[3], −20[4]. Evaluation of methylation status in a promoter region of p15, p16 and MGMT genes showed biallelic hypermethylation pattern of 5′ promoter region only in MGMT gene. PC-MDS is the first t-MDS derived cell line, and based on its immunological, cytogenetic and molecular characterization could be a new tool in evaluation of complex biology of MDS and a model for methylation studies.

Abnormal Localization of Neutrophil Elastase in Patients with Severe Congenital Neutropenia.

Mutations in the ELA2 gene encoding neutrophil elastase (NE) in patients with severe congenital neutropenia (SCN) are involved in the pathogenesis of this disorder, possibly due to the abnormal protein trafficking and accelerated apoptosis of myeloid cells. In this study we precisely examined the localization of NE in neutrophils and myeloid precursor cells in bone marrow in patients with SCN using immunofluorescence microscopy equipped with three-dimensional analysis program. Three patients with SCN were enrolled in this study. All patients with SCN showed heterozygous mutation in the ELA2 gene. In normal subjects the pattern of localization of NE in mature neutrophils was almost similar to those of myeloperoxidase (MPO), proteinase 3, lysosomal associated membrane protein 2 (LAMP2). Administration of G-CSF to normal subjects did not affect the pattern of the localization of these proteins in neutrophils. In contrast, mature neutrophils elicited by the administration of G-CSF in patients with SCN NE predominantly localized to the plasma membranes. A small part of NE was detected in the cytoplasmic compartment. The pattern of localization of NE was significantly different from those of MPO, proteinase 3, and LAMP2 in SCN patients, suggesting the abnormal traffic of NE to granules. Adaptor proteins 3 (AP3) specifically shuttles transmembrane cargo proteins from the trans-Golgi to lysosomes. AP3 of myeloid progenitor cells enriched for CD33-positive cells in normal bone marrow was localized in both cytoplasm and plasma membranes. The localization pattern of AP3 was completely consistent with those of NE, MPO, and LAMP2. The localization of AP3 of promyelocytes in patients with SCN was observed in both plasma membranes and cytoplasm. This finding was completely similar to that in normal myeloid precursor cells. However, the localization of NE of promyelocytes in SCN patients was predominantly in plasma membrane. The figures merged apparently presented the different localization of NE and AP3. This result was confirmed by the 3-dimensional analysis with histogram. The localizations of other constituents of primary granules, MPO, poteinase 3, and LAMP2, were consistent with those of AP3. These observations suggest that the mislocalization of NE in myeloid precursor cells in SCN patients does not result from a generalized impairment of protein trafficking but is specific to the mutant NE. The abnormal localization of NE may be involved in the pathogenesis of SCN associated with ELA2 mutation.

Ribavirin-Induced Dysplasia.

Ribavirin is a synthetic nucleoside analogue given with pegylated interferon alfa-2a for the treatment of hepatitis C. Hemolytic anemia has been reported in 10–15% of patients, usually within 1–2 weeks of the initiation of therapy. The pathogenesis of this phenomenon is unclear. Pegylated interferon alfa-2a has been associated with pancytopenia. We report two cases of ribavirin-induced anemia that are not due solely to hemolysis.Case A is a 56 year old African-American man, status post liver transplant in July 2005 for hepatitis C. He had a recurrence in February 2006, and was placed on ribavirin 1200 mg daily and pegylated interferon 1.8 micrograms weekly. Within 1 month, his hemoglobin dropped to 8.1 g/dL, his MCV was 107mm3 and his reticulocyte count was 57K per mm3, while maintaining a normal white blood cell and platelet count. His Coombs test was negative. There was no response to the administration of erythropoietin. Bone marrow aspirate and biopsy showed marked erythroid dysplasia - including multi-nucleated precursors and megaloblastosis. There was no morphological evidence of myeloid or megakaryocytic dysplasia. Cytogenetic studies revealed a normal male karyotype (46XY).Case B is a 58 year old African-American man with hepatitis C. In January 2006 he was started on ribavirin 1200 mg daily, in combination with pegylated interferon. Eight weeks later, he was found to have a hemoglobin of 8.1 g/dl - decreased from 12.2 g/dL. The MCV was 104.4 mm3, and the reticulocyte count was 56K per mm3. His Coombs test was negative. Bone marrow biopsy performed at that time showed similar findings to Case A. Ribavirin and pegylated interferon were discontinued; a repeat bone marrow after six weeks showed only minimal dyserythropoiesis. He is currently maintaining his hemoglobin between 12 and 13 g/dL, with no transfusion or erythropoietin support.There are no reported associations between ribavirin and erythroid dysplasia in humans. Ribavirin has been shown to have cytotoxic effects on bone marrows in animal models. In one study, ribavirin was given intraperitoneally to rats in doses ranging from 10 to 200 mg/kg 1. Bone marrow samples obtained at 24 and 48 hours demonstrated micronuclei in erythroid precursors, with recovery at 72 hours. Rhesus monkeys given ribavirin at doses of 30 or 100 mg/kg/day intramuscularly for 10 days developed a normocytic, normochromic anemia, which was severe in the high-dose group 2. The bone marrows demonstrated a marked decrease in late erythroid precursors, while the early precursors remained unchanged. There was also vacuolization of the erythroid precursors as well as erythrophagocytosis. No changes were noted in the myeloid precursor cells, and the megakaryocytes appeared increased. After discontinuation of treatment the hemoglobin returned to normal, demonstrating a reversible effect.The bone marrows of the patients demonstrate ineffective erythropoiesis with marked erythroid dysplasia and megaloblastosis. These findings are associated with a decreased proportion of cells in the DNA synthesis of the cell cycle, an increase in the fraction of late precursor cells undergoing apoptosis, and an interruption in mitosis. These findings may be due to ribavirin's interference in the maturation of erythroid cells. These observations need to be confirmed in a prospective manner in a large cohort of patients; they also suggest the need for a more complete evaluation of anemia in patients treated with ribavirin.

SGN-33 Modulates Cytokine and Chemokine Production by Activated Monocytes and Macrophages.

SGN-33, a humanized IgG1 anti-CD33 antibody that targets CD33, a sialoadhesion family member expressed on myeloid precursor cells, macrophages, and monocytes, is currently in Phase I clinical trials for acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). To further characterize and define the mechanism of action of SGN-33, we undertook a series of experiments in AML cell lines and in cultures of primary normal human monocytes and macrophages. SGN-33 has previously been shown to eliminate tumor cells through antibody-mediated effector functions including Antibody-Dependent Cellular Cytotoxicity (ADCC) and Complement Dependent Cytotoxicity (CDC). We have now demonstrated that SGN-33 binding also initiates Antibody-Dependent Cellular Phagocytosis (ADCP). Because CD33 has been hypothesized to mediate signal transduction, we explored the biochemical effects of SGN-33 binding on AML cell lines and normal monocytes and macrophages. SGN-33 binding stimulated tyrosine phosphorylation of CD33 and recruitment of SHP-1, with maximal effects between 5 to 30 minutes. Initiation of signaling by SGN-33 was not dependent upon antibody crosslinking. Furthermore, SGN-33 exposure significantly reduced the syntheses of pro-inflammatory cytokines (TNF-a, IL-6, IL-1b) and chemokines (RANTES, MCP-1, IL-8) by macrophages cultured in the presence of tumor cell conditioned media. Production of these cytokines by monocytes and macrophages activated in vitro by IFN-g and TGF-b were also blocked by SGN-33 binding (up to 100% reduction in TNF-a levels in vitro). These data demonstrate that the mechanisms of action of anti-CD33 immunotherapy may be broader than previously known. Because pro-inflammatory cytokines have been implicated in the growth and survival of tumor cells in patients, the capacity of SGN-33 to block these factors may yield clinical benefit. This hypothesis is currently being evaluated in clinical studies of SGN-33.