Search

Immunotherapy, especially immune checkpoint blockade therapy, represents a major milestone in the history of cancer therapy. However, the current response rate to immunotherapy among cancer patients must be improved; thus, new strategies for sensitizing patients to immunotherapy are urgently needed. Erythroid progenitor cells (EPCs), a population of immature erythroid cells, exert potent immunosuppressive functions. As a newly recognized immunosuppressive population, EPCs have not yet been effectively targeted. In this review, we summarize the immunoregulatory mechanisms of EPCs, especially for CD45 + EPCs. Moreover, in view of the regulatory effects of EPCs on the tumor microenvironment, we propose the concept of EPC-immunity, present existing strategies for targeting EPCs, and discuss the challenges encountered in both basic research and clinical applications. In particular, the impact of existing cancer treatments on EPCs is discussed, laying the foundation for combination therapies. The aim of this review is to provide new avenues for improving the efficacy of cancer immunotherapy by targeting EPCs., (© 2024 UICC.)

Introduction: CD8 + T cell tolerance plays an important role in tumor escape. Recent studies have shown that CD45 + erythroid progenitor cells (CD45 + EPCs) generated through splenic extramedullary erythropoiesis suppress tumor immunity. However, the mechanism underlying how CD45 + EPCs mediate CD8 + T cell tolerance remains incompletely understood and requires further research., Methods: In this study, the antigen-processing abilities of CD45 + EPCs was verified through both in vitro and in vivo experiments. We have used the method of co-culture in vitro and adoptive transfer experiments in vivo to explore the effects of CD45 + EPCs on CD8 + T cell tolerance. RNA-sequencing analysis and blocking experiments were used to evaluate the role of ROS in the CD45 + EPC mediated tolerance of CD8 + T cells. Finally, we incorporated uric acid into the adoptive transfer experiments to rescue the CD45 + EPC mediated tumor-promoting effect., Results and Discussion: We found that CD45 + EPCs take up soluble proteins, present antigenic epitopes on their surface, and induce antigen-specific CD8 + T cell anergy. In addition, we found that CD45 + EPC directly nitrates tyrosine within the TCR/CD8 complex via the production of reactive oxygen species and peroxynitrite, preventing CD8 + T cells from responding to their specific peptide antigens. Furthermore, uric acid treatment effectively abolished the immunosuppressive effects of CD45 + EPCs during CD8 + T cell adoptive transfer, thereby enhancing the anti-tumor efficacy. These results demonstrated that CD8 + T cell tolerance in tumor-bearing mice is induced by CD45 + EPCs. The results of this study have direct implications for tumor immunotherapy., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Fan, Peng, Wang, Sun, Dong, Zhou, Chen and Huang.)

SARS-CoV-2 infection is associated with lower blood oxygen levels, even in patients without hypoxia requiring hospitalization. This discordance illustrates the need for a more unifying explanation as to whether SARS-CoV-2 directly or indirectly affects erythropoiesis. Here, we show significantly enriched CD71 + erythroid precursors/progenitors in the blood circulation of COVID-19 patients. We found that these cells have distinctive immunosuppressive properties. In agreement, we observed a strong negative correlation between the frequency of these cells with T and B cell proportions in COVID-19 patients. The expansion of these CD71 + erythroid precursors/progenitors was negatively correlated with the hemoglobin levels. A subpopulation of abundant erythroid cells, CD45 + CD71 + cells, co-express ACE2, TMPRSS2, CD147, and CD26, and these can be infected with SARS-CoV-2. In turn, pre-treatment of erythroid cells with dexamethasone significantly diminished ACE2/TMPRSS2 expression and subsequently reduced their infectivity with SARS-CoV-2. This provides a novel insight into the impact of SARS-CoV-2 on erythropoiesis and hypoxia seen in COVID-19 patients., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Newborns are highly susceptible to infectious diseases. The underlying mechanism of neonatal infection susceptibility has generally been related to their under-developed immune system. Nevertheless, this notion has recently been challenged by the discovery of the physiological abundance of immunosuppressive erythroid precursors C D71 + e rythroid c ell s (CECs) in newborn mice and human cord blood. Here, as proof of concept, we show that these cells are also abundant in the peripheral blood of human newborns. Although their frequency appears to be more variable compared to their counterparts in mice, they rapidly decline by 4 weeks of age. However, their proportion remains significantly higher in infants up to six months of age compared to older infants. We found CD45 expressing CECs, as erythroid progenitors, were the prominent source of reactive oxygen species (ROS) production in both humans and mice. Interestingly, a higher proportion of CD45 + CECs was observed in the spleen versus bone marrow of neonatal mice, which was associated with a higher ROS production by splenic CECs compared to their siblings in the bone marrow. CECs from human newborns suppressed cytokine production by CD14 monocytes and T cells, which was partially abrogated by apocynin in vitro . Moreover, the depletion of CECs in neonatal mice increased the number of activated effector immune cells in their spleen and liver, which rendered them more resistant to Listeria monocytogenes infection. This was evident by a significant reduction in the bacteria load in the spleen, liver and brain of treated-mice compared to the control group, which enhanced their survival rate. Our finding highlights the immunoregulatory processes mediated by CECs in newborns. Thus, such tightly regulated immune system in newborns/infants may explain one potential mechanism for the asymptomatic or mild COVID-19 infection in this population., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Elahi, Vega-López, Herman-Miguel, Ramírez-Estudillo, Mancilla-Ramírez, Motyka, West and Oyegbami.)

Steady-state erythropoiesis generates new erythrocytes at a constant rate, and it has enormous productive capacity. This production is balanced by the removal of senescent erythrocytes by macrophages in the spleen and liver. Erythroid homeostasis is highly regulated to maintain sufficient erythrocytes for efficient oxygen delivery to the tissues, while avoiding viscosity problems associated with overproduction. However, there are times when this constant production of erythrocytes is inhibited or is inadequate; at these times, erythroid output is increased to compensate for the loss of production. In some cases, increased steady-state erythropoiesis can offset the loss of erythrocytes but, in response to inflammation caused by infection or tissue damage, steady-state erythropoiesis is inhibited. To maintain homeostasis under these conditions, an alternative stress erythropoiesis pathway is activated. Emerging data suggest that the bone morphogenetic protein 4 (BMP4)-dependent stress erythropoiesis pathway is integrated into the inflammatory response and generates a bolus of new erythrocytes that maintain homeostasis until steady-state erythropoiesis can resume. In this perspective, we define the mechanisms that generate new erythrocytes when steady-state erythropoiesis is impaired and discuss experimental models to study human stress erythropoiesis., (Copyright © 2020 ISEH -- Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.)

Anti-helminth responses require robust type 2 cytokine production that simultaneously promotes worm expulsion and initiates the resolution of helminth-induced wounds and hemorrhaging. However, how infection-induced changes in hematopoiesis contribute to these seemingly distinct processes remains unknown. Recent studies have suggested the existence of a hematopoietic progenitor with dual mast cell-erythrocyte potential. Nonetheless, whether and how these progenitors contribute to host protection during an active infection remains to be defined. Here, we employed single cell RNA-sequencing and identified that the metabolic enzyme, carbonic anhydrase (Car) 1 marks a predefined bone marrow-resident hematopoietic progenitor cell (HPC) population. Next, we generated a Car1-reporter mouse model and found that Car1-GFP positive progenitors represent bipotent mast cell/erythrocyte precursors. Finally, we show that Car1-expressing HPCs simultaneously support mast cell and erythrocyte responses during Trichinella spiralis infection. Collectively, these data suggest that mast cell/erythrocyte precursors are mobilized to promote type 2 cytokine responses and alleviate helminth-induced blood loss, developmentally linking these processes. Collectively, these studies reveal unappreciated hematopoietic events initiated by the host to combat helminth parasites and provide insight into the evolutionary pressure that may have shaped the developmental relationship between mast cells and erythrocytes., Competing Interests: Mark C. Siracusa is the founder and president of NemaGen Discoveries.

Renal anemia in chronic kidney disease is treated with recombinant human erythropoietin (rhEPO). However, some patients with anemia do not respond well to rhEPO, emphasizing the need for a more biocompatible EPO. Differentiation protocols for hepatic lineages have been modified to enable production from human induced pluripotent stem cell (hiPSC)-derived EPO-producing cells (EPO cells). However, markers for hiPSC-EPO cells are lacking, making it difficult to purify hiPSC-EPO cells and therefore to optimize EPO production and cell counts for transplantation. To address these issues, we investigated whether CD140b and CD73 could be used as markers for hiPSC-EPO cells. We measured the expression of EPO, CD140b, and CD73 in hiPSC-EPO cells and the EPO concentration in the cell supernatant by immunohistochemistry and enzyme-linked immunosorbent assays on culture day 13, revealing that expression levels of CD140b and CD73 are correlated with the level of EPO. In addition, rates of CD140b + CD73 + cells were observed to be correlated with the concentration of EPO. Thus, our results suggest that CD140b and CD73 may be markers for hiPSC-EPO cells., (© 2020 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.)

Inflammation alters bone marrow hematopoiesis to favor the production of innate immune effector cells at the expense of lymphoid cells and erythrocytes. Furthermore, proinflammatory cytokines inhibit steady-state erythropoiesis, which leads to the development of anemia in diseases with chronic inflammation. Acute anemia or hypoxic stress induces stress erythropoiesis, which generates a wave of new erythrocytes to maintain erythroid homeostasis until steady-state erythropoiesis can resume. Although hypoxia-dependent signaling is a key component of stress erythropoiesis, we found that inflammation also induced stress erythropoiesis in the absence of hypoxia. Using a mouse model of sterile inflammation, we demonstrated that signaling through Toll-like receptors (TLRs) paradoxically increased the phagocytosis of erythrocytes (erythrophagocytosis) by macrophages in the spleen, which enabled expression of the heme-responsive gene encoding the transcription factor SPI-C. Increased amounts of SPI-C coupled with TLR signaling promoted the expression of Gdf15 and Bmp4 , both of which encode ligands that initiate the expansion of stress erythroid progenitors (SEPs) in the spleen. Furthermore, despite their inhibition of steady-state erythropoiesis in the bone marrow, the proinflammatory cytokines TNF-α and IL-1β promoted the expansion and differentiation of SEPs in the spleen. These data suggest that inflammatory signals induce stress erythropoiesis to maintain erythroid homeostasis when inflammation inhibits steady-state erythropoiesis., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Background: Antibody screening in pretransfusion tests is necessary to avoid critical complications of blood transfusion. Although red blood cells (RBCs) expressing relevant alloantigen(s) have been used for serologic antibody screening, little attention has been given to the use of cell lines, in which blood group antigen gene(s) are transduced, as reagent RBCs for antibody screening., Study Design and Methods: The use of an erythroid progenitor cell line for serologic tests was studied. The expression of blood group antigens of erythroid progenitor cells was analyzed by genotyping and flow cytometry. Serologic analysis including hemagglutination was performed using erythroid progenitor cells to evaluate their sensitivity for antibody detection. Overexpression of exogenous erythroid antigen by lentiviral transduction was carried out and investigated for antibody detection sensitivity., Results: Erythroid progenitor cells contained a substantial amount of hemoglobin and expressed sufficient levels of blood group antigens to detect corresponding monoclonal antibodies. Furthermore, the cell line could acquire an exogenous RBC antigen after lentiviral transduction and detected corresponding monoclonal and alloantibodies with equal sensitivity to antigen-positive RBCs., Conclusion: Application of erythroid progenitor cell lines for screening for unexpected antibodies could be helpful in solving issues such as reagent availability associated with the conventional RBC-based assay. The genetic expandability of erythroid progenitor cell lines by gene modification techniques could lead to the development of more convenient reagent RBCs., (© 2018 AABB.)

Impaired immunity in patients with late-stage cancer is not limited to antitumor responses, as demonstrated by poor vaccination protection and high susceptibility to infection 1-3 . This has been largely attributed to chemotherapy-induced impairment of innate immunity, such as neutropenia 2 , whereas systemic effects of tumors on hematopoiesis and adoptive immunity remain incompletely understood. Here we observed anemia associated with severe deficiency of CD8 + T cell responses against pathogens in treatment-naive mice bearing large tumors. Specifically, we identify CD45 + erythroid progenitor cells (CD71 + TER119 + ; EPCs) as robust immunosuppressors. CD45 + EPCs, induced by tumor growth-associated extramedullary hematopoiesis, accumulate in the spleen to become a major population, outnumbering regulatory T cells (T reg s) and myeloid-derived suppressor cells (MDSCs). The CD45 + EPC transcriptome closely resembles that of MDSCs, and, like MDSCs, reactive oxygen species production is a major mechanism underlying CD45 + EPC-mediated immunosuppression. Similarly, an immunosuppressive CD45 + EPC population was detected in patients with cancer who have anemia. These findings identify a major population of immunosuppressive cells that likely contributes to the impaired T cell responses commonly observed in patients with advanced cancer.

NOTCH signaling is required for the arterial specification and formation of hematopoietic stem cells (HSCs) and lympho-myeloid progenitors in the embryonic aorta-gonad-mesonephros region and extraembryonic vasculature from a distinct lineage of vascular endothelial cells with hemogenic potential. However, the role of NOTCH signaling in hemogenic endothelium (HE) specification from human pluripotent stem cell (hPSC) has not been studied. Here, using a chemically defined hPSC differentiation system combined with the use of DLL1-Fc and DAPT to manipulate NOTCH, we discover that NOTCH activation in hPSC-derived immature HE progenitors leads to formation of CD144 + CD43 - CD73 - DLL4 + Runx1 + 23-GFP + arterial-type HE, which requires NOTCH signaling to undergo endothelial-to-hematopoietic transition and produce definitive lympho-myeloid and erythroid cells. These findings demonstrate that NOTCH-mediated arterialization of HE is an essential prerequisite for establishing definitive lympho-myeloid program and suggest that exploring molecular pathways that lead to arterial specification may aid in vitro approaches to enhance definitive hematopoiesis from hPSCs.

Background: Pure red cell aplasia (PRCA) is an uncommon disease which involves an almost complete absence of the erythroid lineage in bone marrow (BM) and causes severe anemia. Cases due to monoclonal gammopathy occurring in plasma cell disorder have been infrequently reported. Here we report a case of PRCA associated plasma cell disorder, especially monoclonal gammopathy of undetermined significance (MGUS)., Methods: A 55-year-old male visited the ER due to general weakness. At his initial visit he exhibited severe anemia. Mild intravascular hemolysis was suspected. For anemia evaluation, BM examination was performed. In BM aspiration, almost no erythroid precursor cells were observed. Also, plasma cells were relatively elevated, at 7.2%. Serum electrophoresis and immunofixation revealed paraproteinemia of 5.1 g/L (IgG and lambda). No hypercalcemia, renal insufficiency or lytic bone lesions were found. This unusual case showed MGUS accompanied by PCRA. We were also able to assume the erythroid cell-specific restriction due to paraprotein, because we ruled out possible causes of PRCA., Results: We discovered several reported cases associated with plasma cell dyscrasia. However, most of these cases involved plasma cell myeloma, characterized by high immunoglobulin burden. Our case demonstrates that PRCA is also observed in cases with MGUS, where immunoglobulin burden is low., Conclusions: It is not yet accurately known, what parts of erythroid precursors are targeted by M-protein nor what the mechanism is. Therefore, additional research into this matter is necessary.

The reaction of Hector Battifora mesothelial epitope-1 (HBME-1) antibody with scattered pronormoblasts in normal bone marrow core biopsy specimens has been reported. This study evaluated the immunohistochemical profile of HBME-1 in a panel of 52 normal, dyserythropoietic and neoplastic marrow samples. We compared the staining property of HBME-1 with that of the commonly used erythroid marker, glycophorin A (CD235a) and in each case, we semi-quantitatively evaluated the HBME-1/CD235a-positive cells ratio. In normal samples, HBME-1 labelled scattered immature erythroid precursors. In dyserythropoietic specimens, HBME-1 stained nucleated erythroid precursors in varying degrees, from pronormoblast through normoblast stages, with the highest intensity in immature forms. Overall, the cellular background of non-erythroid progenitors, erythrocytes and neoplastic cells did not react with HBME-1, except in leukaemia cases with myelodysplasia-related changes. Our study shows that HBME-1 is a useful marker to identify immature erythroid precursors and that an HBME-1/CD235a-positive cells ratio ≥10% is associated with dyserythropoiesis., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/)

Hemolytic disease of the newborn (HDN) arising from MNSs incompatibility is rare, with few reports of prolonged anemia and reticulocytopenia following HDN. We report the younger of 2 male siblings, both of whom had anti-M-induced HDN and anemia persisting for over a month. Peripheral reticulocytes remained inappropriately low for the degree of anemia, and they needed multiple red cell transfusions. Viral infections were ruled out. Corticosteroids were given for suspected pure red cell aplasia. Anemia and reticulocytopenia subsequently improved. Colony-forming unit erythroid assay revealed erythropoietic suppression of M antigen-positive erythroid precursor cells cultured with maternal or infant sera containing anti-M. In conclusion, maternal anti-M caused HDN and prolonged anemia by erythropoietic suppression in 2 siblings.

Background and Objectives: Previously, a weak phenotype Am or Bm was assumed to be caused by a reduction of A or B gene expression in bone marrow cells, but not in mucus-secreting cells. However, ABO expression has not been examined in erythroid progenitor cells of Am or Bm individuals., Materials and Methods: We carried out in vitro erythroid differentiation of CD34(+) cells from peripheral blood of a Bm individual harbouring a 3.0-kb deletion including an erythroid cell-specific regulatory element, named the +5.8-kb site, in intron 1 of the human ABO blood group gene., Results: During the in vitro differentiation of CD34(+) cells from this Bm individual into erythroid cells, B-antigens were not detectable on the cultured cells by flow cytometric analysis, and allele-specific RT-PCR consistently detected the transcripts from the O allele, but not from the B allele. Moreover, chromatin immunoprecipitation assay demonstrated that both RUNX1 and GATA-2 or GATA-1 were bound to the +5.8-kb site in cultured erythroid cells expressing ABO., Conclusion: It is likely that the +5.8-kb site enhances transcription from the ABO promoter in erythroid cells through binding of RUNX1 and GATA-2 or GATA-1., (© 2014 International Society of Blood Transfusion.)

Parvovirus B19 (B19V) is a small non-enveloped DNA virus of the Parvoviridae family. It is an obligate human pathogen that preferentially replicates in erythroid progenitor cells. B19V is the causative agent of multiple erythroid-related diseases due to replication-induced cytotoxicity. Despite its strong erythroid tropism and related acute disease association, B19V has been determined to persist in many other non-erythroid tissues. This review summarizes and appraises what is known about concomitant B19V DNA persistence and non-acute viral gene expression in various, particularly non-erythroid, tissue types. The methods utilized for B19V detection are described, focusing on the discrepancies in outcomes among the employed assays. The studies where investigations focused on the impact of persistent B19V expression on cellular signaling pathways are also summarized. These studies demonstrate the expanse of the types of cells capable of in vivo B19V expression as well as the possible effect of persistent viral infection on the cellular microenvironment. Overall, these reports indicate that B19V commonly persists in a wide range of both erythroid and non-erythroid tissues, and that low-level viral gene expression can be detected in some persistently infected cells. B19V capsid RNA or proteins have been reported in bone marrow, colon, heart, liver, lymphoid, synovial, testicular, and thyroid tissues. In a sub-set of these cases, B19V capsid mRNA or proteins have been associated with increased inflammatory-related gene expression. The development of standard protocols to assay for B19V infection and expression in the context of non-erythroid, non-acute disease is warranted, and with further targeted studies, may begin to elucidate the impact of persistent B19V infection in vivo. These studies may determine the most conducive cellular environment for persistent gene expression and possible impact on disease pathogenesis., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Background: Kell is a complex blood group system comprising 35 antigens. Kell antigens are absent from rare red cells of the Ko (null) phenotype and expressed only weakly in the Kmod phenotype. Molecular analysis of three uncommon KEL alleles elucidated the obscure pattern of inheritance of Kell antigens in one family., Materials and Methods: Standard serological methods were employed. All exons, flanking intronic sequence and introns 15 and 16 of KEL were sequenced from genomic DNA. cDNA was obtained from erythroid cells cultured from progenitor cells isolated from peripheral blood., Results: The Kmod propositus was heterozygous for two KEL mutations: c.2107G>A, p.Gly703Arg and a synonymous mutation, c.1719C>T, in the codon for p.573Gly. Sequencing of cDNA revealed that c.1719C>T caused skipping of exon 16, resulting in a silent allele. Her KEL:3,-4 brother was heterozygous for KEL*03/04 and c.1719C/T., Conclusion: A synonymous mutation caused complete exon skipping, despite being located 16 bases downstream of the 3′ splice site, resulting in a null KEL allele. The combined effects of two mod alleles, one responsible for KEL3 expression and the other for p.Gly703Arg, were probably responsible for an unexpected KEL:3,-4 phenotype.

We performed a series of experiments under a working hypothesis that cross-linked oligomers of ribosomal protein S19 (RP S19) play an essential role in definitive erythropoiesis as a ligand of the C5a receptor of erythroblasts and macrophages. We found molecules functionally and immunologically indistinguishable from RP S19 oligomers in the extracellular fluid of porcine and guinea pig bone marrow. When an increased hematopoietic state was induced in guinea pigs by bloodletting, the bone marrow RP S19 oligomer concentration was concomitantly increased. However, when the RP S19 oligomers were immunologically neutralized or the C5a receptor was pharmacologically antagonized, hyper-erythropoiesis induced by bloodletting was prevented and the anemic state was retarded in guinea pigs. When the RP S19 oligomers were neutralized in mice after bloodletting, the reactive hyper proliferation of erythroblasts in the spleen was prevented. Proerythroblasts and erythroblasts prepared by bone marrow aspiration from healthy individuals were found to express significant levels of the C5a receptor and type 2 transglutaminase genes. Majority of erythroblasts in cord blood of healthy newborns bore the C5a receptor. Taken together, these results support our hypothesis., (© 2013.)

The peripheral blood Pig-a assay has shown promise as a tool for evaluating in vivo mutagenicity. In this study five laboratories participated in a collaborative trial that evaluated the transferability and reproducibility of a rat Pig-a assay that uses a HIS49 antibody reacts with an antigen found on erythrocytes and erythroid progenitors. In preliminary work, flow cytometry methods were established that enabled all laboratories to detect CD59-negative erythrocyte frequencies (Pig-a mutant frequencies) of <10×10(-6) in control rats. Four of the laboratories (the in-life labs) then treated male rats with a single oral dose of N-nitroso-N-ethylurea, 7,12-dimethylbenz[a]anthracene (DMBA), or 4-nitroquinoline-1-oxide (4NQO). Blood samples were collected up to 4 weeks after the treatments and analyzed by flow cytometry for the frequency of CD59-negative cells among total red blood cells (RBCs; RBC Pig-a assay). RBC Pig-a assays were conducted in the four in-life laboratories, plus a fifth laboratory that received blood samples from the other laboratories. In addition, three of the five laboratories performed a Pig-a assay on reticulocytes (RETs; PIGRET assay), using blood from the rats treated with DMBA and 4NQO. The four in-life laboratories detected consistent, time- and dose-related increases in RBC Pig-a mutant frequency (MF) for all three test articles. Furthermore, comparable results were obtained in the fifth laboratory that received blood samples from other laboratories. The three laboratories conducting the PIGRET assay also detected consistent, time- and dose-related increases in Pig-a MF, with the RET MFs increasing more rapidly with time than RBC MFs. These results indicate that rat Pig-a assays using a HIS49 antibody were transferable between laboratories and that data generated by the assays were reproducible. The findings also suggest that the PIGRET assay may detect the in vivo mutagenicity of test compounds earlier than the RBC Pig-a assay., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Donor lymphocyte infusion (DLI) using unstimulated leukapheresis is one of the most effective treatment strategies for patients with hematological malignancies; its graft-versus-leukemia effects make it especially effective in chronic myeloid leukemia patients who relapsed after allogeneic stem cell transplantation (allo-HSCT). However, DLI application is limited by the development of graft-versus-host disease and aplasia, and thus cannot be routinely applied for prophylaxis. Therefore, important questions remain to be answered, such as when, and whom to DLI? Recent advances enable DLI using allografts of granulocyte colony-stimulating factor-mobilized peripheral blood progenitor cells; allodepleted donor T cells; and infusions of donor-derived, ex vivo-expanded, CD8(+) cytotoxic T lymphocyte, which can decrease relapse and improve transplant outcomes. Preemptive immunotherapy of relapse was also introduced based on the determination of mixed chimerism and minimal residual disease. In this review, we summarize the latest developments in recent strategies that will affect future DLI efficacy - focusing on the disadvantages and advantages of each protocol for the treatment, preemptive therapy, and prophylaxis of relapse., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The prevalence, clinical associations and pathogenic role of newly identified autoantibodies to the erythropoietin receptor (EPOR) in patients with anaemia were investigated. Sera from 203 patients with immune-related or chronic kidney diseases were screened for anti-EPOR antibodies by enzyme-linked immunosorbent assay, and antibody specificity was evaluated by immunoprecipitating EPOR from AS-E2 cells using purified immunoglobulin (Ig) fractions. In addition, the pathogenic role of anti-EPOR antibodies was determined by examining their inhibitory effects on AS-E2 cell proliferation. Clinical findings were compared between patients with and without anti-EPOR antibodies, in all patients and those with systemic lupus erythematosus (SLE). Serum anti-EPOR antibodies were detected in 52 patients. Purified IgG or IgM fractions from anti-EPOR antibody-positive sera immunoprecipitated EPOR and inhibited the EPO-dependent proliferation of AS-E2 cells in a dose-dependent manner. Anti-EPOR antibodies were associated with low haemoglobin concentrations and reticulocytopenia in all patients enrolled and those with SLE. Further, there was a negative correlation between the levels of anti-EPOR antibodies and the number of bone marrow erythroblasts in patients who underwent bone marrow examinations. These findings suggest that EPOR autoantibodies are present in a subset of patients with anaemia and that impaired erythropoiesis can be mediated by anti-EPOR antibodies, which functionally neutralize EPO activity., (© 2012 Blackwell Publishing Ltd.)

Natural killer (NK) cells function as early effector cells in the innate immune defense against viral infections and also participate in the regulation of normal and malignant hematopoiesis. NK cell activities have been associated with early clearance of viremia in experimental simian immunodeficiency virus and clinical human immunodeficiency virus type 1 (HIV-1) infections. We have previously shown that NK cells function as major cytotoxic effector cells in vaccine-induced immune protection against Friend virus (FV)-induced leukemia, and NK cell depletion totally abrogates the above protective immunity. However, how NK cells recognize retrovirus-infected cells remains largely unclear. The present study demonstrates a correlation between the expression of the products of retinoic acid early transcript-1 (RAE-1) genes in target cells and their susceptibility to killing by NK cells isolated from FV-infected animals. This killing was abrogated by antibodies blocking the NKG2D receptor in vitro. Further, the expression of RAE-1 proteins on erythroblast surfaces increased early after FV inoculation, and administration of an RAE-1-blocking antibody resulted in increased spleen infectious centers and exaggerated pathology, indicating that FV-infected erythroid cells are recognized by NK cells mainly through the NKG2D-RAE-1 interactions in vivo. Enhanced retroviral replication due to host gene-targeting resulted in markedly increased RAE-1 expression in the absence of massive erythroid cell proliferation, indicating a direct role of retroviral replication in RAE-1 upregulation.

The CD34(+) compartment of grafts for clinical allogeneic hematopoietic cell transplantation (HCT) is very heterogeneous. It contains hematopoietic stem cells and several different progenitor cell populations. This study assesses (1) the content of these populations in clinical grafts from G-CSF-mobilized PBMCs, BM and cord blood, (2) the functional correlation of the graft composition with time to engraftment of neutrophils, platelets and reticulocytes and (3) donor age-related changes. Quantitative flow cytometry showed that the distribution of the progenitor subsets differed significantly between the graft sources and that donor age-related changes occur. In patients after myeloablative allogeneic HCT, accelerated platelet and reticulocyte engraftment correlated with the content of common myeloid and/or megakaryocyte erythroid progenitors in the graft. These findings show that a better understanding of the progenitor compartment in human hematopoietic grafts could lead to improved strategies for the development of cellular therapies, for example in situations where platelet engraftment is delayed.

Signal transduction mediated by Fas-associated death domain protein (FADD) represents a paradigm of coregulation of apoptosis and cellular proliferation. During apoptotic signaling induced by death receptors including Fas, FADD is required for the recruitment and activation of caspase 8. In addition, a death receptor-independent function of FADD is essential for embryogenesis. In previous studies, FADD deficiency in embryonic stem cells resulted in a complete lack of B cells and dramatically reduced T cell numbers, as shown by Rag1(-/-) blastocyst complementation assays. However, T-specific FADD-deficient mice contained normal numbers of thymocytes and slightly reduced peripheral T cell numbers, whereas B cell-specific deletion of FADD led to increased peripheral B cell numbers. It remains undetermined what impact an FADD deficiency has on hematopoietic stem cells and progenitors. The current study analyzed the effect of simultaneous deletion of FADD in multiple cell types, including bone marrow cells, by using the IFN-inducible Mx1-cre transgene. The resulting FADD mutant mice did not develop lymphoproliferation diseases, unlike Fas-deficient mice. Instead, a time-dependent depletion of peripheral FADD-deficient lymphocytes was observed. In the bone marrow, a lack of FADD led to a dramatic decrease in the hematopoietic stem cells and progenitor-enriched population. Furthermore, FADD-deficient bone marrow cells were defective in their ability to generate lymphoid, myeloid, and erythroid cells. Thus, the results revealed a temporal requirement for FADD. Although dispensable during lymphopoiesis post lineage commitment, FADD plays a critical role in early hematopoietic stages in the bone marrow.

Objective: These studies were performed to test the hypothesis that DNMT1 is required for maintenance of DNA methylation and repression of the γ-globin gene in adult-stage erythroid cells., Materials and Methods: DNMT1 levels were reduced by nucleofection of small interfering RNA targeting DNMT1 in chemical inducer of dimerization-dependent multipotential mouse bone marrow cells containing the human β-globin gene locus in the context of a yeast artificial chromosome and in primary cultures of erythroid progenitor cells derived from CD34(+) baboon bone marrow cells. The effect of reduced DNMT1 levels on globin gene expression was measured by real-time polymerase chain reaction and the effect on globin chain synthesis in primary erythroid progenitor cell cultures was determined by biosynthetic radiolabeling of globin chains followed by high-performance liquid chromatography analysis. The effect on DNA methylation was determined by bisulfite sequence analysis., Results: Reduced DNMT1 levels in cells treated with siDNMT1 were associated with increased expression of γ-globin messenger RNA, an increased γ/γ+β chain ratio in cultured erythroid progenitors, and decreased DNA methylation of the γ-globin promoter. Similar effects were observed in cells treated with decitabine, a pharmacological inhibitor of DNA methyltransferase inhibitor., Conclusions: DNMT1 is required to maintain DNA methylation of the γ-globin gene promoter and repress γ-globin gene expression in adult-stage erythroid cells., (Copyright © 2011 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.)

The presence of intrauterine inflammation has been associated with adverse neurologic outcomes in preterm infants, but the precise mechanisms of fetal brain injury remain unclear. We sought to evaluate inflammatory cell trafficking, fetal organ damage, and molecular regulation in the fetoplacental unit using an established mouse model of preterm birth associated with intrauterine inflammation. Gestational sacs were harvested 6 hours after intrauterine infusion of saline or lipopolysaccharide (LPS). Histologic, immunohistochemical, and molecular investigations were performed to identify target organ damage and the cellular phenotype of inflammatory cells and to quantify circulating inflammatory and hematopoietic mediators within the placental and fetal tissue. There was widespread increase in fetal macrophages in LPS-exposed pups, including within the leptomeninges of the brain, associated with significantly higher of interleukin 6 levels in LPS-exposed pups. Although no specific central nervous system injury (necrosis or apoptosis) was documented, liver hematomas were seen significantly more frequently in LPS-exposed pups. Circulating nucleated fetal erythrocytes were also present more frequently with LPS exposure without significantly higher erythropoietin levels than saline-exposed mice. The presence of increased macrophages, increased circulating interleukin 6 levels, and increased circulating erythroid precursors in LPS-exposed pups suggests that these are significant factors associated with potential target organ damage, such as liver hematomas, associated with intrauterine inflammation and preterm birth. The role of macrophages within the fetal leptomeninges is unclear, but they may play an important role in inflammatory-mediated brain damage, and further investigation of their significance and potential as therapeutic targets is warranted.

Antigen presentation, a key mechanism in immune responses, involves two main signals: the first is provided by the engagement of a major histocompatibility complex (MHC), class I or class II, with their TCR receptor in lymphocytes, whereas the second demands the participation of different co-stimulatory molecules, such as CD28, CTLA-4 and their receptors B7.1 and B7.2. Specific T-cell activation and deactivation are achieved through this signalling. The aim of our study is to characterise, in the acute experimental autoimmune encephalomyelitis (EAE) model in Lewis rat, the temporal expression pattern of these molecules as well as the cells responsible for their expression. To accomplish that, MBP-immunised female Lewis rats were daily examined for the presence of clinical symptoms and sacrificed, according to their clinical score, at different phases during EAE. Spinal cords were cut with a cryostat and processed for immunohistochemistry: MHC-class I and MHC-class II, co-stimulatory molecules (B7.1, B7.2, CD28, CTLA-4) and markers of dendritic cells (CD1 for immature cells and fascin for mature cells). Our results show that microglial cells are activated in the inductive phase and, during this phase and peak, they are able to express MHC-class I, MHC-class II and CD1, but not B7.1 and B7.2. This microglial phenotype may induce the apoptosis or anergy of infiltrated CD28+ lymphocytes observed around blood vessels and in the parenchyma. During the recovery phase, microglial cells express high MHC-class I and class II and, those located in the surroundings of blood vessels, displayed the B7.2 co-stimulatory molecule. These cells are competent to interact with CTLA-4+ cells, which indicate an active role of microglial cells in modulating the ending of the immune response by inducing lymphocyte activity inhibition and Treg activation. Once clinical symptomatology disappeared, some foci of activated microglial cells (MHC-class II+/B7.2+) were still present in concomitance with CTLA-4+ cells, suggesting a prolonged involvement of microglia in lymphocyte inhibition and tolerance promotion. In addition to microglia, during the inductive and recovery phases, we also found perivascular ED2+ cells and fascin+ cells which are able to migrate to the parenchyma and may play a role in lymphocytic regulation. Further studies to understand the specific function played by these cells are warranted., (Copyright 2010 Elsevier B.V. All rights reserved.)

Pro-inflammatory cytokines like interferon-gamma (IFN-gamma) are considered to be important in the development of anaemia of chronic disease (ACD). Both, inhibitory and stimulatory activities of IFN-gamma on erythropoiesis have been observed in vitro earlier. IFN-gamma induces several biochemical pathways in human monocytes, among them neopterin formation by GTP-cyclohydrolase I (GTP-CH I) and tryptophan degradation by the enzyme indoleamine 2,3-dioxygenase (IDO). IDO-mediated tryptophan deprivation efficiently inhibits the growth of proliferating cells and microbes, thus we wanted to examine whether enhanced tryptophan degradation by monocytic precursor cells also suppresses erythropoiesis. Therefore, IFN-gamma-mediated pathways were investigated in human CD34(+) progenitor cells, and effects of IFN-gamma on the proliferative activity of different progenitor subpopulations were studied. Cells were either cultivated in agar-conditioned medium (ACM) or in medium containing erythroid growth factors interleukin-3 (IL-3) and stem cell factor (SCF; EGFCM). Stimulation of CD34(+) cells with IFN-gamma in different doses (either 5000U/ml once or 200 and 400U/ml every other day) induced tryptophan degradation and in parallel also neopterin formation. Unstimulated cells cultured with ACM produced higher amounts of neopterin and kynurenine (all p<0.05). IFN-gamma stimulated higher kynurenine and neopterin formation in cells cultivated in EGFCM, stimulation with 400U IFN-gamma every other day was most effective. IFN-gamma stimulated the growth and proliferation of CFU-E and BFU-E (3-8) in both media. In conclusion, stimulation of haematopoietic stem cells with IFN-gamma activates IDO and neopterin formation, and it also exerts an influence on the proliferation of various stem cell populations., (Copyright 2009 Elsevier GmbH. All rights reserved.)

Background: Exercise in humans augments the mobilization of circulating hematopoietic progenitor cells (CD34(+)) from the bone marrow. We investigated the effect of inflammation on erythroid marrow activity by mobilization of erythroid progenitor cells (EPs) along with soluble markers of erythropoiesis., Methods: Ten healthy athletes who participated in an ultradistance foot race participated in the study. Peripheral blood mononuclear cells were isolated, before (phase I), at the end (phase II), and at 48 h post-race (phase III). EPs were detected as burst colony forming units (BFU-e) and colonies were scored at day 14. Markers of inflammation (C-reactive protein, serum amyloid-A, interleukin-6, ferritin and S100B) and bone marrow activity (erythropoietin, soluble transferrin receptor and lipocalin-2) were assessed., Results: An approximately three-fold decrease in BFU-e number was observed at phase II. sTfR concentrations were also decreased at phase II and remained decreased at phase III. However, EPO and lipocalin-2 concentrations reached a maximum value at phase II, with a tendency to decrease at phase III., Conclusions: These findings indicate that exercise-induced inflammation modulates bone marrow homeostasis leading to an increase in leukocyte turnover and a decrease in erythroid compartment. It appears that lipocalin-2 is the main factor that regulates the production and mobilization of EPs.

Erythropoietin (Epo) binds and activates the Epo receptor (EpoR) on the surface of erythroid progenitor cells resulting in formation of erythrocytes. Recently, EpoR was reported to be expressed on non-erythroid cells suggesting a role for Epo outside of erythropoiesis. However those studies employed antibodies with questionable specificity and the significance of the observations are controversial. In order to accurately determine the expression of EpoR proteins in cells, we have generated a panel of novel anti-human EpoR monoclonal antibodies. One of these antibodies (A82) was particularly sensitive and it detected the EpoR protein on intact cells by flow cytometry and by western blot analysis with cell lysates. Both methods were optimized and using them, EpoR protein was detected by western immunoblotting with lysates from fewer than 200 EpoR positive control cells and the positive signals were proportional to EpoR protein expression level with a minimal signal in EpoR negative cells. The proteins detected by western blot analysis using A82 included full-length EpoR ( approximately 59kDa) as well as smaller EpoR fragments derived from the EPOR gene. These results indicate that A82 can be used to examine low level EpoR expression in cells by western and flow cytometry allowing an improved understanding of EpoR expression and metabolism., (Copyright 2009 Elsevier B.V. All rights reserved.)

Thalassemic children are at a high risk of graft rejection in cord blood transplantation. To investigate this possible mechanism, the authors evaluated the effect of panel reactive antibody on the growth of CD34(+) cells in vitro. On semisolid medium, CD34(+) cells derived from cord blood were incubated with thalassemic serum, and colony-forming units were counted after 10 days of culture. After incubation with serum-specific panel reactive antibody, profound decreases were found in the numbers of CFU-GM, CFU-GEMM and BFU-E compared with controls. The results indicated that serum-specific panel reactive antibody might have an apparent inhibition effect on proliferation and differentiation of cord blood CD34(+) cells.

The Gerbich (Ge) antigens are a collection of high-incidence antigens carried on the red blood cell membrane glycoproteins, glycophorins C and D. Antibodies against these antigens are uncommon, and there have been only rare case reports of hemolytic disease of the fetus and newborn due to anti-Ge. In this case report, we present a neonate with severe anemia and hyperbilirubinemia due to anti-Ge3. Routine and special laboratory studies undertaken in this case suggested two mechanisms for the patient's hemolysis and persistent anemia. Antibody-dependent hemolysis was associated with early-onset hyperbilirubinemia, anemia, and a mild reticulocytosis, and inhibition of erythroid progenitor cell growth was associated with late anemia and normal bilirubin and reticulocyte values. Though rare, anti-Ge3 can be a dangerous antibody in pregnancy. Affected neonates may require intensive initial therapy and close follow-up for at least several weeks after delivery.

The pathogenic parvovirus B19 (B19V) has an extreme tropism for human erythroid progenitor cells. In vitro, only a few erythroid leukemic cell lines (JK-1 and KU812Ep6) or megakaryoblastoid cell lines (UT7/Epo and UT7/Epo-S1) with erythroid characteristics support B19V replication, but these cells are only semipermissive. By using recent advances in generating large numbers of human erythroid progenitor cells (EPCs) ex vivo from hematopoietic stem cells (HSCs), we produced a pure population of CD36(+) EPCs expanded and differentiated from CD34(+) HSCs and assessed the CD36(+) EPCs for their permissiveness to B19V infection. Over more than 3 weeks, cells grown in serum-free medium expanded more than 800,000-fold, and 87 to 96% of the CD36(+) EPCs were positive for globoside, the cellular receptor for B19V. Immunofluorescence (IF) staining showed that about 77% of the CD36(+) EPCs were positive for B19V infection, while about 9% of UT7/Epo-S1 cells were B19V positive. Viral DNA detected by real-time PCR increased by more than 3 logs in CD36(+) EPCs; the increase was 1 log in UT7/Epo-S1 cells. Due to the extensive permissivity of CD36(+) EPCs, we significantly improved the sensitivity of detection of infectious B19V by real-time reverse transcription-PCR and IF staining 100- and 1,000-fold, respectively, which is greater than the sensitivity of UT7/Epo-S1 cell-based methods. This is the first description of an ex vivo method to produce large numbers of EPCs that are highly permissive to B19V infection and replication, offering a cellular system that mimics in vivo infection with this pathogenic human virus.

Erythropoietin (EPO) is a growth factor, regulating the proliferation and differentiation of erythroid progenitor cells. In this study, we generated five monoclonal antibodies (mAbs) that reacted specifically with recombinant human EPO (rhEPO). Epitope exclusion and other experiments showed that the mAbs obtained were divided into two groups, differing in recognition sites for rhEPO: group 1 mAbs recognize the N-terminal region of rhEPO, whereas group 2 mAbs seem to recognize a conformation-dependent epitope. Although most of the previously reported anti-EPO antibodies that recognized the N-terminal region of EPO lacked the EPO-neutralizing activity, the group 1 mAbs obtained here had the rhEPO-neutralizing activity. Therefore, the group 1 mAbs may be useful for future study on structure-function relationship of EPO. One of the group 2 mAbs, 5D11A, showed the highest affinity for rhEPO with K(D) value 0.52 nM and had the highest rhEPO-neutralizing activity. Using this mAb, we developed a reproducible and sensitive enzyme-linked immunosorbent assay for the quantification of bioactive rhEPO.

In order to study the role of the cytokine interleukin-3 (IL-3) and its signaling pathways in erythropoiesis of beta-thalassemia/HbE erythroid progenitor cells, CD34 positive cells were isolated from peripheral blood of patients and healthy subjects. After culturing the cells in the presence or absence of IL-3, cell viability was measured by trypan blue staining and apoptotic cells were analyzed by flow cytometry. After 7 days of culture the highest percent erythroid progenitor cell viability was obtained with cells from healthy subjects, while the lowest percentage was found in those from splenectomized beta-thalassemia/HbE. Viability of beta-thalassemia/HbE erythroid progenitor cells in the presence of IL-3 was higher than that of nonsupplemented cells. In addition, specific inhibitors of protein kinase C (Ro-318220), phospholipase C (U-73122) and Janus kinase 2 (AG-490) were used to investigate the involvement of signaling pathways in erythropoiesis. Percent apoptosis of erythroid progenitor cells from splenectomized beta-thalassemia/HbE subjects treated with RO-318220 was higher than those of nonsplenectomized beta-thalassemia/HbE and healthy subjects. Treatment with U-73122 resulted in enhanced percent apoptotic cells from normal and beta-thalassemia/HbE subjects. All these effects were independent of IL-3 treatment.

Acute myeloid leukemia (AML) is a clonal disease characterized by heterogeneous involvement of hematopoietic stem cell/progenitor cell populations. Using FLT3 internal tandem duplication (FLT3/ITD) as a molecular marker, we tested the hypothesis that clinical outcome in AML correlates with disease involvement of CD34(+)/CD33(-) precursors. Diagnostic specimens from 24 children with FLT3/ITD-positive AML were sorted by fluorescence-activated cell sorting (FACS), and resultant CD34(+)/CD33(-) and CD34(+)/CD33(+) progenitors were analyzed directly and after colony-forming cell (CFC) assay for the presence of FLT3/ITD. FLT3/ITD was present in all CD34(+)/CD33(+) patient samples. In contrast, FLT3/ITD was detected in CD34(+)/CD33(-) progenitors in only 19 of 24 samples. A bipotent progenitor was affected in a subset of patients, as evidenced by the presence of FLT3/ITD in both granulocyte-macrophage colony-forming unit (CFU-GM) and erythroid burst-forming unit (BFU-E) colonies. Those patients in whom CD34(+)/CD33(-) precursors harbored the FLT3/ITD had worse clinical outcome; actuarial event-free survival (EFS) at 4 years from study entry for those patients with and without FLT3/ITD detection in CD34(+)/CD33(-) progenitors was 11% +/- 14% versus 100% +/- 0%, respectively (P = .002). This study suggests that FLT3/ITD involvement in CD34(+)/CD33(-) precursors is heterogeneous and that detection of the mutation in the less-mature progenitor population may be associated with disease resistance.

A novel stem cell marker prominin-1 (CD133) has been shown to be expressed on a subpopulation of CD34(+) haematopoietic stem and progenitor cells. The aim of this study was to compare in parallel commercially available CD34(+) and CD133(+) isolation methods based on paramagnetic bead-coupled antibodies using clinical-grade samples of mobilized peripheral blood from 10 individual healthy donors under identical conditions. The CD133 negative fraction from the first selection was used for CD34(+) enrichment to obtain an additional CD34(+)/CD133(-) population. Although no significant difference in total cell expansion between cells isolated from the three procedures was observed in a 7-day cytokine-driven suspension culture, the long-term culture-initiating cell assay demonstrated that cells derived by CD34(+) isolation contain less primitive progenitors than those isolated based on CD133(+) selection. Interestingly, CD34(+)-enriched progenitors, especially the CD34(+)/CD133(-) fraction, contained a significantly higher proportion of erythroid colony-forming cells, whereas the highest content of myeloid colony-forming cells was concentrated in the CD133(+) selected cells. These subtle differences between CD34(+) and CD133(+) immunomagnetic selection will have to be explored for their potential clinical relevance.

The pathogenesis of malarial anemia is multifactorial, and the mechanisms responsible for its high mortality are poorly understood. Studies indicate that host mediators produced during malaria infection may suppress erythroid progenitor development (Miller, K.L., J.C. Schooley, K.L. Smith, B. Kullgren, L.J. Mahlmann, and P.H. Silverman. 1989. Exp. Hematol. 17:379-385; Yap, G.S., and M.M. Stevenson. 1991. Ann. NY Acad. Sci. 628:279-281). We describe an intrinsic role for macrophage migration inhibitory factor (MIF) in the development of the anemic complications and bone marrow suppression that are associated with malaria infection. At concentrations found in the circulation of malaria-infected patients, MIF suppressed erythropoietin-dependent erythroid colony formation. MIF synergized with tumor necrosis factor and gamma interferon, which are known antagonists of hematopoiesis, even when these cytokines were present in subinhibitory concentrations. MIF inhibited erythroid differentiation and hemoglobin production, and it antagonized the pattern of mitogen-activated protein kinase phosphorylation that normally occurs during erythroid progenitor differentiation. Infection of MIF knockout mice with Plasmodium chabaudi resulted in less severe anemia, improved erythroid progenitor development, and increased survival compared with wild-type controls. We also found that human mononuclear cells carrying highly expressed MIF alleles produced more MIF when stimulated with the malarial product hemozoin compared with cells carrying low expression MIF alleles. These data suggest that polymorphisms at the MIF locus may influence the levels of MIF produced in the innate response to malaria infection and the likelihood of anemic complications.

This study was designed to investigate the regulatory role of soluble interleukin-6 receptor (sIL-6R) and interleukin-6 (IL-6) fusion protein (Hyper-IL-6) in the differentiation of human myeloid and erythroid progenitors by a serum-free liquid suspension culture system, using the human cord blood-derived CD34(+)CD38(-) cells as a target. We found that Hyper-IL-6 promoted the generation of CD15(+) granulocytic and CD14(+) monocytic cells and suppressed that of CD14(-)CD1a(+) dendritic cells from CD36(-)CD15(-)CD14(-)CD1a(-)IL-6R(+) myeloid progenitors. Conversely, CD34(+)CD38(-) cell-derived early erythroid progenitors were negative for IL-6R expression. Hyper-IL-6 potentiated the generation of CD36(+)glycophorinA(high) mature erythroid cells from the IL-6R(-) early erythroid progenitors. Our results indicate that Hyper-IL-6 augments the generation of CD15(+) granulocytic, CD14(+) monocytic and CD36(+)glycophorinA(high) cell and suppresses that of CD14(-)CD1a(+) dendritic cells.

Human acute myeloid leukemias with the t(8;21) translocation express the AML1-ETO fusion protein in the hematopoietic stem cell compartment and show impairment in erythroid differentiation. This clinical finding is reproduced in multiple murine and cell culture model systems in which AML1-ETO specifically interferes with erythroid maturation. Using purified normal human early hematopoietic progenitor cells, we find that AML1-ETO impedes the earliest discernable steps of erythroid lineage commitment. Correspondingly, GATA-1, a central transcriptional regulator of erythroid differentiation, undergoes repression by AML1-ETO in a nonconventional histone deacetylase-independent manner. In particular, GATA-1 acetylation by its transcriptional coactivator, p300/CBP, a critical regulatory step in programming erythroid development, is efficiently blocked by AML1-ETO. Fusion of a heterologous E1A coactivator recruitment module to GATA-1 overrides the inhibitory effects of AML1-ETO on GATA-1 acetylation and transactivation. Furthermore, the E1A-GATA-1 fusion, but not wild-type GATA-1, rescues erythroid lineage commitment in primary human progenitors expressing AML1-ETO. These results ascribe a novel repressive mechanism to AML1-ETO, blockade of GATA-1 acetylation, which correlates with its inhibitory effects on primary erythroid lineage commitment.

With approximately 2.4 billion people at risk, Plasmodium falciparum malaria infection caused by an infectious bite of an Anopheles mosquito continues to be a major cause of mortality and morbidity, mainly in the tropics and subtropics. Measures to control the mosquito vector on a broad scale are expensive and need to be maintained continuously. The rapid emergence of parasite strains that are resistant to affordable drugs highlights the urgent need for a cheap and effective vaccine. Candidate vaccines that have been developed to date target different stages of the parasite life cycle. This review describes the recent advances in the development of a vaccine that aims to terminate the infection at its first stage in the liver. The candidate vaccines that are currently under clinical evaluation are introduced and the results from recent trials discussed. The review aims to explain the immunologic challenges a successful vaccine has to meet, as well as the different strategies that are currently employed in an attempt to induce a protective immune response. Furthermore, an outline of available options to be tested in the near future will be presented.

The destruction of erythrocytes and defects in erythropoiesis are among the most frequently observed causes of morbidity in severe Plasmodium falciparum malaria. The molecular mechanisms involved remain unclear, despite extensive investigation. We show here, for the first time, that tagging with the parasite rhoptry protein ring surface protein 2 (RSP2) is not restricted to the surfaces of normal erythrocytes, as previously reported, but that it extends to erythroid precursor cells in the bone marrow of anemic malaria patients. Monoclonal mouse antibodies and human sera from patients with severe anemia, reacting with RSP2-tagged erythrocytes, induced cell destruction by phagocytosis and complement activation in vitro. Our observations reveal a new parasite mechanism implicated in the destruction of normal erythrocytes and probably dyserythropoiesis in malaria patients. These data suggest that the tagging of host cells with RSP2 may trigger anemia in falciparum malaria.

Background: Kell and XK, two distinct red blood cell membrane proteins, are linked by a disulfide bond and form the Kell blood group complex. Kell surface antigens are expressed early during erythropoiesis but the onset of expression of XK which carries the Kx antigen is unknown., Study Design and Methods: To determine whether Kell and XK are synchronously expressed, sorted human hematopoietic progenitor cells and mouse progenitor cells of defined lineage were studied. To determine the onset of expression, human marrow and cord blood cells were sorted into three subpopulations, representing stem, multipotent, and erythroid progenitor cells, and the expression of Kell and XK was determined by reverse transcription-polymerase chain reaction (RT-PCR) and fluorescence-activated cell sorting (FACS) analysis. Mouse Kell and XK transcripts were determined by cDNA blotting of progenitor cells of defined lineage., Results: By RT-PCR, human peripheral blood progenitor cells had weak expression of Kell and XK transcripts but FACS analysis did not detect surface antigens. Kell and XK transcripts are expressed in multipotent progenitor cells and these cells express Kell surface antigens. The expression of Kx antigen in progenitor cells was not determined owing to nonspecific reactions with the antibody. By cDNA blotting, mouse Kell expression was detected in bipotential megakaryocytes-erythroid cells and in colony-forming units-erythroid (CFU-E) and burst-forming units-erythroid (BFU-E), whereas XK was only detected in CFU-E and BFU-E., Conclusion: Both Kell and XK transcripts occur early during erythropoiesis; however, expression may not be coincident because, in mice, Kell transcripts, but not XK, occur in bipotential megakaryocytes-erythroid progenitor cells.

Background: The Cord Blood Transplantation (COBLT) Study banking program was initiated in 1996. The study goals were to develop standard operating procedures for cord blood (CB) donor recruitment and banking and to build an ethnically diverse unrelated CB bank to support a transplantation protocol., Study Design and Methods: The hematopoietic progenitor cell (HPC) and lymphocyte subset (LS) content of approximately 8000 CB units were characterized, and these results were correlated with donor ethnicity, birth weight, gestational age, sex, and type of delivery., Results: There was a significant correlation of CD34+ cell count with colony-forming unit (CFU)-granulocyte-macrophage (r=0.68, p<0.001), CFU-granulocyte-erythroid-macrophage-megakaryocyte (r=0.52, p<0.001), burst-forming unit-erythroid (BFU-E; r=0.61, p<0.001), and total CFUs (r=0.67, p<0.001). Nucleated red blood cell count was significantly correlated with total CD34+ (r=0.56, p<0.001), total CFU (r=0.50, p<0.001), BFU-E (r=0.48, p<0.001), and counts of CD34+ subsets (p<0.001). Caucasian ethnicity was significantly correlated with higher CD3+/CD4+, CD19+, and CD16+/CD56+ LSs. Furthermore, CD34+/CD38- and CD34+/CD61+ CB units (HPC-C) were significantly lower in African American and Asian persons compared to Caucasian and Hispanic persons. Male sex was associated with significantly fewer CD3+/CD4+, CD19+, and CD16+/CD56+ but increased CD3+/CD8+ LSs (p<0.001). Finally, cesarean section was associated with significantly higher total CFU and CD16+/CD56+ but lower CD3+/CD4+, CD3+/CD8+, and CD19+ LSs., Conclusion: These results provide a standard and range for uniformly processed HPC-C progenitor cells and LSs. CB progenitor cells and/or LSs may in the future predict for rapidity of engraftment, incidence of graft-versus-host disease, speed and quality of immunore- constitution, graft-versus-tumor effects, and/or success of gene transfection after CB transplantation.

There have been no reports of severe haemolytic disease of the newborn (HDN) due to Gerbich (Ge) antibodies. Two babies with HDN due to anti-Ge3, both born to the same mother, are described. The anti-Ge appeared in the first pregnancy and was not detectable in the first trimester, the babies' reticulocyte and bilirubin values were not greatly elevated (similar to HDN due to Kell antibodies), and the anaemia in both cases was either not apparent or not severe until 2 to 4 weeks after birth. Ge antigens are found on glycophorins (GPs) C and D; GPC, like Kell, has been shown to be expressed early on erythroid progenitor cells. The maternal anti-Ge3 was shown to promote phagocytosis of Ge+ early erythroid progenitors by monocytes (similar to what has been reported with anti-K and K+ progenitor cells). Thus, anti-Ge3 may cause immune destruction of erythroid progenitors and possibly suppression of erythropoiesis (which would explain the reticulocyte and bilirubin values seen in both cases). Anti-Ge3 appears to be capable of causing severe HDN. We suggest that babies born to mothers with anti-Ge should have their haemoglobin concentrations monitored for signs of anaemia for several weeks after birth. Functional assays may prove useful.

In order to gain more insight into mechanisms operating on the haematopoietic activity of the T-cell-derived cytokine, interleukin-17 (IL-17) and target cells that first respond to its action in vivo, the influence of a single intravenous injection of recombinant mouse IL-17 on bone marrow progenitors, further morphologically recognizable cells and peripheral blood cells was assessed in normal mice up to 72 h after treatment. Simultaneously, the release of IL-6, IL-10, IGF-I, IFN-gamma and NO by bone marrow cells was determined. Results showed that, in bone marrow, IL-17 did not affect granulocyte-macrophage (CFU-GM) progenitors, but induced a persistant increase in the number of morphologically recognizable proliferative granulocytes (PG) up to 48 h after treatment. The number of immature erythroid (BFU-E) progenitors was increased at 48 h, while the number of mature erythroid (CFU-E) progenitors was decreased up to 48 h. In peripheral blood, white blood cells were increased 6 h after treatment, mainly because of the increase in the number of lymphocytes. IL-17 also increased IL-6 release and NO production 6 h after administration. Additional in vitro assessment on bone marrow highly enriched Lin- progenitor cells, demonstrated a slightly enhancing effect of IL-17 on CFU-GM and no influence on BFU-E, suggesting the importance of bone marrow accessory cells and secondary induced cytokines for IL-17 mediated effects on progenitor cells. Taken together, these results demonstrate that in vivo IL-17 affects both granulocytic and erythroid lineages, with more mature haematopoietic progenitors responding first to its action. The opposite effects exerted on PG and CFU-E found at the same time indicate that IL-17, as a component of a regulatory network, is able to intervene in mechanisms that shift haematopoiesis from the erythroid to the granulocytic lineage.

The domestic cat is an excellent model for the development of therapeutic protocols that target hematopoietic stem cells (HSCs) because it is relatively resistant to complications related to bone marrow transplantation. To identify a plentiful source of HSC that could be used as targets for gene transduction and transplantation, the livers of 28 mid-gestation fetuses (28-52 days) and late-gestation fetuses (53 days-term) were analyzed for erythroid, myeloid, lymphoid, and uncommitted hematopoietic progenitor cells by flow cytometry. We found that the fetal liver mononuclear cells (FLMCs) contained 57% erythroid progenitors during mid-gestation, but this percentage declined to 43% as gestation progressed. Myelomonocytic cells within FLMC were more numerous in late-gestation (31%) than in mid-gestation (18%). Two monoclonal antibodies (mAb), CH 152 and CH 755, which recognize cells with the potential to reconstitute multilineage hematopoiesis in cats, were tested. Approximately, 32% of FLMC from late-gestation fetuses expressed the epitope recognized by mAb CH 152, a significant increase above the 12% positive cells in mid-gestation fetuses. Approximately, 33% of hepatic mononuclear cells expressed the epitope recognized by mAb CH 755 in both mid-term and late-term fetuses. When expressed in absolute numbers, medians of 2.7 x 10(7) CH 152-positive cells and 3.2 x 10(7) CH 755-positive cells were extracted from the late-term fetal livers of individual cats. T-lymphocytes were a minor component (<3%) of FLMC, despite their presence in the thymus and spleen. These data suggest that the late-term feline fetal liver is a suitable source of mutipotential hematopoietic cells that could be used for gene therapy protocols in the cat., (Copyright 2004 Elsevier B.V.)

Despite normal levels of erythropoiesis in PU.1(-/-) embryos, PU.1(-/-) fetal hematopoietic progenitors are unable to establish sustained erythropoiesis in the adult bone marrow. This study demonstrates that PU.1(-/-) fetal erythroid progenitors are synergistically expanded by TPO plus SCF, but not combinations of EPO plus SCF, IL-3 or GM-CSF. The EPO defect is not corrected by a constitutively active variant of EPOR. Microarray analysis identified several candidate PU.1 target genes known to affect cytokine signaling and gene regulation in the erythroid lineage. These data suggest that PU.1 plays an important role in regulating the proliferation of immature erythroid progenitors.

Human parvovirus B19 (B19) infection causes human bone marrow failure, by affecting erythroid-lineage cells which are well-known target cells for B19. The anaemia induced by B19 infection is of minor clinical significance in healthy children and adults, however, it becomes critical in those afflicted with haemolytic diseases. This condition is called transient aplastic crisis, and the pathogenesis is explained by the short life-span of red blood cells. Similarly, fetuses are thought to be severely affected by B19-intrauterine infection in the first and second trimester, as the half-life of red blood cells is apparently shorter than RBC at the bone marrow haematopoietic stage. On the other hand, B19 is also the causative agent of persistent anaemia in immunocompromised patients, transplant recipients and infants. The deficiencies of appropriate immune responses to B19 impair viral elimination in vivo, which results in enlargement of B19-infected erythroid-lineage cells. The B19-associated damage of erythroid lineage cells is due to cytotoxicity mediated by viral proteins. B19-infected erythroid-lineage cells show apoptotic features, which are thought to be induced by the non-structural protein, NS1, of B19. In addition, B19 infection induces cell cycle arrests at the G(1) and G(2) phases. The G(1) arrest is induced by NS1 expression prior to apoptosis induction in B19-infected cells, while the G(2) arrest is induced not only by infectious B19 but also by UV-inactivated B19, which lacks the ability to express NS1. In this review, we address the clinical manifestations and molecular mechanisms for B19-induced anaemia in humans and a mouse model, and of B19-induced cell cycle arrests in erythroid cells., (Copyright 2003 John Wiley & Sons, Ltd.)