bluuebunny/crossref_metadata_2025 huggingface dataset:
NOD Hematopoietic Stem Cells Exhibit Autonomous Behavior and a Competitive Advantage in Allogeneic Recipients That Maps to the Idd9 Locus.
Authors: Chilton Paula M. , Rezzoug Francine , Ratajczak Janina , Ratajczak Mariusz , Huang Yiming , Tanner Michael K. , Fugier-Vivier Isabelle J. , Ildstad Suzanne T.
Published: April 2020

Multiple hematopoietic defects have been defined in NOD mice and in humans with type I diabetes, including defects in myeloid cells and antigen presenting cells that correlate with diabetes progression. Since the replacement of HSC in NOD mice can eliminate the progression of autoimmunity and control on-going autoimmune responses, we characterized the function of HSC from NOD mice. We found that purified HSC from NOD mice have an autonomous behavior when transplanted in allogeneic recipient strains as reflected by significantly enhanced engraftment in allogeneic recipients. NOD HSC were able to compete for engraftment with syngeneic HSC even when the NOD and syngeneic HSC were given at a 1:1 ratio. NOD BMC produced a higher number of splenic colonies compared to B10.BR BMC in the allogeneic day 12 CFU-S assay. We also demonstrated that NOD HSC had a high resistance to irradiation, as reflected by the cell survival 20 hours after irradiation and in the in vitro CFC assay. These data suggest that NOD HSC escape alloreactivity and compete with normal HSC. The enhanced engraftment ability in allogeneic recipients of NOD HSC was not due to an increase in frequency of primitive HSC, enumerated by day 35 cobblestone area forming cells (CAFC). This finding was further confirmed by the fact that there was no difference in the long-term repopulating cell phenotype (CD49e+/CD49ddim) between HSC obtained from NOD, B10.BR or C57BL/10 mice. Notably, NOD bone marrow cells exhibit significantly enhanced chemotaxis to SDF-1 in vitro and significantly increased HSC adhesion to primary stroma. This was associated with an increase in the expression of VCAM-1, ICAM-1 and ICAM-2 on NOD HSC. Using NOD mice congenic at selected Idd loci with C57BL/10, we determined that the enhanced engraftment potential of NOD HSC mapped to the Idd9 (insulin-dependent diabetes) locus and, as such, the TNF receptor family as well as ski/sno genes may be involved in the mechanism underlying the autonomy of NOD HSC. In conclusion, NOD HSC exhibit increased autonomy in vivo and in vitro compared to non-diabetic strains, and engraft better in allogeneic recipients, possibly due to enhanced migration and adherence to the microenvironment. This finding may be of interest for a better understanding of disease pathogenesis and in developing cell-based strategies to cure diabetes.
Ex Vivo Production of Transfusable Red Blood Cells at Large Scale from Cord Blood CD34+ Cells Using a Coculture System with Human Telomerase Catalytic Subunit (hTERT)-Transfected Human Stromal Cells at Early Phase and with Acitivated Macrophage from Cord Blood CD34+ Cells at Late Phase.
Authors: Fujimi Akihito , Matsunaga Takuya , Kobune Masayoshi , Kawano Yutaka , Tanaka Ikuta , Takemoto Naofumi , Akiyama Takehide , Hamada Hirofumi , Niitsu Yoshiro
Published: April 2020

New sources of red blood cells (RBC) would improve the transfusion capacity of blood centers. Several investigators have previously reported that erythroblasts could be obtained from hematopoietic stem cells including those of cord blood (CB) by in vitro culture. However, transfusion of erythroblasts may not be suitable for supplementation of acute blood loss because it should need some time lag until hemoglobin (RBC) boost in circulation due to the fact that transfused erythroblasts once lodged at bone marrow where they undergo maturation into RBCs which are bound to be released into circulation. We have developed a culture system for producing large quantity of enucleated RBCs (e-RBCs) as well as erythroblasts from CB in vitro: one unit e-RBCs (2 x 1012 RBCs) was obtained from one standard CB unit (corresponding to 2 x 106 CD34+ cells) using a coculture system with hTERT-transfected human stromal cells at early phase followed by with activated macrophage in liquid culture (American Society of Hematology 45th Annual Meeting, SanDiego, 2003). In the present study, we first analyzed the function of those manufactured e-RBCs in comparison of that of adult peripheral blood RBCs (PB-RBCs) or that of eryhthroblasts. The hemoglobin (Hb) content of the e-RBCs quantified by photometric determination was almost equivalent to that of adult PBRBC. A Hb A/Hb F ratio of e-RBC analyzed by high-performance liquid chromatography (HbA: HbF = 35: 65) was between those of CB RBCs (10: 90) and adult PB-RBC (99: 1). Oxygen dissociation curves of e-RBCs measured by Hemox-Analyzer was comparable to that of fresh adult PB-RBCs. The erythroblasts showed adhesive property to stromal cells in vitro but e-RBC did not. When we injected e-RBCs into NOD/SCID mice, they were detectable in circulation while erythroblasts were not. In conclusion, the e-RBCs produced by large-scale culturing system from CB CD34+ cells may be useful for acute blood loss.
New Technology for Red Blood Cell Preservation.
Authors: Tsvetkova Nelly M. , Torok Zsolt , Satpathy Gayana , Banerjee Mitali , Bali Rachna , Dwyre Denis M. , Kheirolomoom Azadeh , Little Erika , Crowe John H. , Tablin Fern
Published: April 2020

A method for freeze-drying red blood cells (RBCs) while maintaining a high degree of viability, has important implications in blood transfusion and clinical medicine. The disaccharide trehalose, found in animals capable of surviving dehydration can aid in this process. We are reporting a method for loading RBCs with trehalose followed by subsequent freeze-drying and rehydration. The loading of erythrocytes is based on the thermal properties of the RBC plasma membranes and provides efficient uptake of the sugar at 37°C in a time span of 7 hours. The data show that RBCs can be loaded with trehalose from the extracellular medium through a combination of osmotic imbalance and the phospholipid phase transition, producing an intracellular trehalose concentration of about 40 mM. Freeze-drying of trehalose loaded RBCs results in water contents in the range between 2 and 4 % and a level of survival of around 37 %, as measured by the extent of hemolysis. Surprisingly, freeze-dried and rehydrated RBCs showed high levels of ATP and 2,3-DPG and low methemoglobin. Biochemical analysis demonstrated that the activities of superoxide dismutase, catalase and acetylcholine esterase in freeze-dried RBCs are similar to those in fresh RBCs. These data provide an important step toward a stable erythrocyte product, which will be invaluable for transfusion and clinical applications. Supported by DARPA grant N66001-03-1-9827
Thromboelastographic (TEG) Characterization of Patients with Hematologic Malignancy and Chemotherapy Induced Thrombocytopenia (CIT).
Authors: Shah Jatin J. , Gomes Marcelo P.V. , Deitcher Steven R.
Published: April 2020

The decision to provide prophylactic platelet transfusions to patients with hematologic malignancy and chemotherapy induced thrombocytopenia (CIT) is often based solely on the platelet count being below a somewhat arbitrary threshold. The ability to rapidly assess the overall (net) coagulation status (thrombotic and hemorrhagic tendencies) of these patients may help limit transfusion to those with a measurable bleeding tendency and reduce overall platelet product consumption. We performed pre- and post-platelet transfusion whole blood thromboelastography (TEG) in 20 pts with leukemia (16) or lymphoma (4) and CIT. Citrated venous blood was obtained prior to and 1 hour after single transfusions of 5 units of random donor platelets. Normal ranges were derived from TEG evaluation of 18 normal controls. Correlation between absolute and % changes in maximum TEG amplitude (MA, mm) and time to initial clot formation (R, min) and absolute and % changes in platelet count was assessed. Pts were defined as normal if MA = 45.7 to 64.9 and R = 8.3 to 19.9; hypocoagulable if MA<45.7 and/or R>19.9; hypercoagulable if MA>64.9 and/or R<8.3; and “mixed” for all other result patterns. Pre-transfusion TEG revealed 17/20 (85%) patients to be hypocoagulable, 1/20 (5%) to be hypercoagulable, and 2/20 (10%) to have a mixed pattern. All hypocoagulable patients had a baseline MA<45.7 and 12/17 had an R>19.9. Pre-transfusion platelet count correlated with baseline MA (r =.47, p=.038). No statistically significant correlation between absolute and % change in platelet count and absolute change in MA, % change in MA, absolute change in R, and % change in R existed. Post transfusion, 17/20(85%) patients had normal MA. Post transfusion, 7/17(41%) hypocoagulable patients had normal TEG, 7/17(41%) remained hypocoagulable, and 3/17(18%) became hypercoagulable. Both “mixed” patients became hypercoagulable; the hypercoagulable patient became normal. 3/5 patients with poor transfusion response (platelet count increase < 10,000/mL) went from hypocoagulable to normal while the remaining 2/5 had MA but not R normalization. Based on our TEG data, 15% of hematologic malignancy patients with CIT did not have a baseline hypocoagulable pattern and may not have needed platelet transfusion. In the 85% who were hypocoagulable at baseline, 41% remained hypocoagulable despite platelet transfusion and may have benefited from additional hemostatic therapy. The patients that normalized did so with variable and at times minimal increases in platelet count. The 18% of baseline hypocoagulable patients that became hypercoagulable post-transfusion may have become transiently prone to thrombosis. Our data supports further evaluation of surrogate markers of overall hemostatic potential, like TEG, as a means of guiding and optimizing platelet transfusion practices.
EB10, an Anti-FLT3 Monoclonal Antibody, Selectively Targets ALL Cell Lines and Primary ALL Blasts without Interfering with Normal Hematopoiesis.
Authors: Piloto Obdulio , Brown Patrick , Li Li , Nguyen Bao , Kim Kyu-Tae , Huso David , Li Yiwen , Li Hongli , Wang Mei-Nai , Witte Larry , Hicklin Daniel J. , Small Donald
Published: April 2020

The class III receptor tyrosine kinase, FLT3, is expressed by >90% of B-lineage acute lymphoblastic leukemias (ALL) blasts. In addition, it is expressed at extremely high levels in ALL patients with MLL-rearrangements or hyperdiploidy and sometimes mutated in these same patients. In this report, we investigated the effects of EB10, an anti-human FLT3 monoclonal antibody capable of preventing binding of FLT3 ligand (FL), on ALL cell lines and primary cells. In vitro studies, examining the ability of EB10 to inhibit FLT3 activation and downstream signaling in ALL cell lines and primary blasts, yielded variable results. In some cell lines FLT3 phosphorylation was inhibited and with it, downstream activation of pathways involving MAPK, AKT, and STAT5 phosphorylation. However, several cell lines actually exhibited FLT3 activation upon antibody treatment, possibly because of antibody-mediated receptor dimerization, and subsequent activation of downstream pathways. Nevertheless, through antibody-mediated cellular cytotoxicity (ADCC) such an antibody could still prove efficacious against leukemia cells in vivo. In fact, EB10 treatment significantly prolongs survival and/or reduces engraftment of ALL cell lines and primary ALL blasts in NOD/SCID mice. This effect might be even more pronounced in a host that was less immune compromised than are NOD/SCID mice. The leukemic cells surviving EB10 treatment in the mice were characterized by FACS analysis and found to express low levels or no FLT3. In contrast to the reduction in engraftment of human ALL primary blasts, EB10 treatment of NOD/SCID mice did not reduce engraftment of human hematopoietic CD34+ cells. Taken together, these data demonstrate that EB10 is selectively cytotoxic to ALL blasts while having little effect on normal hematopoiesis. Such an antibody, either naked or conjugated to radioactive isotopes or cytotoxic agents, may prove useful in the therapy of infant ALL as well as childhood and adult ALL patients whose blasts typically express FLT3.
mTOR Inhibitors Induce Apoptosis and Inhibit Growth of Primary Adult Human ALL in Xenograft and Tissue Culture Models.
Authors: Teachey David T. , Brown Valerie I. , Hall Junior , Cooperman Jonathan , Obzut Dana , Carroll Martin , Choi John K. , Grupp Stephan A.
Published: April 2020

While children with precursor B cell acute lymphoblastic leukemia (ALL) are often cured, adults with ALL usually succumb to their disease. Thus, the development of novel therapeutic agents is paramount. mTOR inhibitors (MTI) are a class of signal transduction inhibitors developed as immunosuppressive agents. We have previously shown the ability of MTI to inhibit growth and induce apoptosis in precursor B ALL cell lines and primary murine pre-B leukemia. To determine if this finding could be translated to clinical therapy, we explored if MTIs would be similarly effective in three primary human adult ALL samples. We have tested the MTIs rapamycin and CCI-779 in these models with similar results, but the results utilizing CCI-779 are presented below.

Stromal culture. ALL blasts were maintained in vitro on irradiated bone marrow stromal cells. Cells normally can be maintained for several weeks in these conditions. Cells were either untreated or treated with CCI-779 at 100ng/ml. Long-term cultures were assessed for the effect of MTI on cell proliferation and short term (48hr) cultures were assessed for induction of apoptosis. Treated cells showed a dramatic decrease in cell proliferation (6–24 fold compared to untreated) and a 4–5 fold increase in apoptotic cells as detected by Annexin-V compared to untreated cells.

NOD/SCID xenografts. To further evaluate the effect of MTI on ALL cells, patient samples were engrafted into NOD/SCID animals for analysis. Robust engraftment, expansion and repopulation in secondary hosts of ALL cells was seen in 78% of tested samples, demonstrating greater than 70% ALL in peripheral blood, bone marrow, and spleen in the majority of the mice. There was greater than 10 fold expansion of disease within the mouse. Engraftment was detected by flow cytometry for human CD19+/CD45+ cells. Engrafted animals with established disease (>5% peripheral blasts) were either not treated or treated with the MTI CCI-779. Currently we have treated 45 mice engrafted from three separate patient samples. Untreated animals continued to show expansion of human ALL cells. In dramatic contrast, animals treated with CCI-779 as a single agent showed a 4–30 fold decrease in peripheral blood blasts and a decrease in splenomegaly (p<.02).

Our data show that both in vitro and in an in vivo model of established ALL, MTIs decrease proliferation of lymphoblasts and promote apoptosis of ALL cells. These results suggest that the mTOR signaling pathway is necessary for the survival of ALL cells and that MTIs should be analyzed as therapeutic agents for the therapy of ALL.
Mutations in the VKORC1 Gene Cause Warfarin Resistance, Warfarin Sensitivity and Combined Deficiency of Vitamin K Dependent Coagulation Factors.
Authors: Oldenburg Johannes , Rost Simone , Fregin Andreas , Geisen Christof , Ivaskevicius Vytautas , Seifried Erhard , Scharrer Inge , Heistinger Maximilian , Tuddenham Edward , Muller-Reible Clemens , Zieger Barbara
Published: April 2020

Coumarins target blood coagulation via inhibition of the vitamin K epoxide reductase multiprotein complex (VKOR). This complex recycles vitamin K 2,3-epoxide to vitamin K hydroquinone, an essential cofactor for the post-translational gamma-carboxylation of several blood coagulation factors. Recently, two groups including ours identified a key component of the VKOR which we named Vitamin K Epoxid Reductase Subunit 1 (VKORC1). The corresponding gene comprises a 5 kb genomic region and consist of three exons encoding a small 163 aa transmembrane protein. Since VKOR was hypothesized to be involved in two heritable diseases, combined deficiency of vitamin K dependent clotting factors type 2 (VKCFD2) and resistance to coumarin-type drugs (warfarin/phenprocoumon resistance, WR) we sequenced the VKORC1 gene in patients with these phenotypes. The same homozygous missense mutation (R98W) was found in two families from Germany and Lebanon with a mild VKCFD2 phenotype. In 7 patients from 5 families with various degrees of WR (weekly warfarin dose ranged from 115 mg to 280 mg, two patients exhibited complete WR) different heterozygous missense mutations distributed throughout the gene (V29L, V45A, R58G, V66M, L128R) were identified. Furthermore, we proved a heterozygous nonsense mutation (W5Stop) in a young female patient with highly increased phenprocoumon sensitivity. Over-expression of the wild-type protein in HEK 293 cells leads to a striking increase in VKOR activity which is sensitive to warfarin inhibition. In contrast to the wild-type protein, the VKCFD2 variant exhibited a dramatically decrease of VKOR activity. The WR variants showed a varying reduction of the VKOR activity, but unexpectedly, were sensitive to warfarin in the HEK cell expression system The reason for the differing WR phenotypes in human and in the HEK cell expression system are still to be elucidated. In conclusion, VKORC1 protein most likely represents the molecular target of coumarins, which have prescribed for oral anticoagulant therapy since more than 60 years. Mutations in the VKORC1 gene are causative for the hereditary conditions of VKCFD2, warfarin resistance and also warfarin sensitivity. Our findings may provide a basis for a rational design of novel anticoagulants targeting VKOR in the future.
Optimal TBI Dose to Improve the Survival in Unrelated Matched Marrow Transplantation for Adult Patients with Severe Aplastic Anemia: Prospective De-Escalation Study of TBI Dose.
Authors: Lee Jong-Wook , Eom Ki Seong , Park Yoon Hee , Kim Yoo Jin , Lee Seok , Min Chang Ki , Kim Hee Je , Kim Dong Wook , Jin Jong Youl , Min Woo Sung , Park Chong Won , Kim Chun Choo
Published: April 2020

Background and Objectives: Patients with severe aplastic anemia (SAA) who do not have suitably HLA-matched related donor generally receive immunosuppressive therapy (IST) as first-line treatment and are considered for transplantation from matched unrelated donor (MUD) if they fail to respond to IST. To determine the optimal total body irradiation (TBI) dose as conditioning regimen in unrelated marrow transplantation for adult SAA patients, we conducted a prospective study to evaluate minimal dose of TBI sufficient to achieve sustained engraftment when it is used in combination with fixed dose of cyclophosphamide (CY).

Methods: Between May 1998 and December 2003 twenty-eight patients were enrolled with a median age of 23 years old (20–44). All patients were multiply transfused and had received 1 or 2 course of intensive IST. Conditioning regimen consisted of CY (120mg/kg) plus TBI and they were divided into 3 group according to the fractionated TBI dose: group 1 (1200 cGy, n=5), group 2 (1000 cGy, n=9), and group 3 (800 cGy, n=14). Donor/recipient pairs were matched for HLA-A, -B by serology and -DRB1 by low-resolution DNA typing (n=15) or HLA-A, -B, and -DRB1 by high-resolution typing (n=13). Patients received CsA+MTx (n=4) or FK506+MTx (n=24) as GVHD prophylaxis.

Results: All patients except one (group 2) achieved sustained engraftment. The incidence of acute GVHD more than grade II was not significantly different (60% in group 1, 38% in group 2, and 43% in group 3), but chronic GVHD developed more frequently in group 1 and 2 (60% & 80%, respectively) compared to group 3 (15%). Patients who received 800 cGy (group 3) had significantly better survival than those 1000 and 1200 cGy (93% in group 3, 44% in group 2, and 40% in group 1; group 3 vs group 1 and 2, p<0.02).

Conclusions: Thus, a TBI dose of 800 cGy in combination with CY (120mg/kg) was sufficient to allow for engraftment in adult patients with SAA who received MUD BMT. Outcome in patients who received 800 cGy of TBI was superior to survival that among patients 1000 and 1200 cGy.
Conversion of Erythropoiesis to Myelopoiesis in Gata1-Deficient Zebrafish Embryos.
Authors: Galloway Jenna L. , Thisse Christine , Zhou Yi , Beltre Rosanna , Thisse Bernard , Zon Leonard I.
Published: April 2020

Primitive hematopoiesis in vertebrates initiates in the embryonic yolk sac and yields nucleated erythrocytes and macrophages that later enter circulation. In zebrafish, a group of blood cells contained within the axial vein make up the intermediate cell mass (ICM), the teleost equivalent of the mammalian yolk sac. To identify novel genes involved in hematopoiesis, a high-throughput whole embryo in situ hybridization screen was performed. Examination of the expression pattern of 3700 clones from an adult zebrafish hematopoietic cDNA library discovered 24 genes with expression in the blood during development. Each stage of hematopoiesis was defined by a subset of genes, providing a molecular signature of blood cell maturation, from hematopoietic progenitors to terminally differentiated erythrocytes. By using antisense morpholinos to the transcription factors gata1 and gata2, we were able to dissect the regulation of these 24 genes. Examination of gene expression in Gata1, Gata2, and Gata1/Gata2-deficient animals revealed that most erythroid genes are dependent upon Gata factors for expression. Surprisingly, three novel genes, expressed in hematopoietic progenitors, do not require Gata factors for their expression demonstrating that some erythroid genes are regulated in a Gata-independent manner. During our analysis, we also found persistent ectopic expression of the myeloid transcription factor, PU.1, in the ICM cells and a subsequent expansion of mpo expressing granulocytes and L-plastin expressing macrophages. By utilizing gata1-GFP transgenic zebrafish, we were able to isolate blood cells by flow cytometry and examine their morphology. We discovered that blood cells from the Gata1-deficient animals exhibited features characteristic of myeloid cells when compared to wild-type blood cells. By confocal microscopy, we detected some blood cells in the ICM of Gata1-deficient embryos that co-express globin and PU.1, while blood cells of wild-type embryos never co-express these markers at this stage. These observations demonstrate that in the absence of Gata1 the presumptive erythroid progenitors have transformed into the myeloid lineage, and that a major cell fate alteration has occurred. Ultimately, our studies have molecularly defined blood development by gene expression, and illustrated that Gata1 governs lineage fate decisions of hematopoietic progenitors in the developing embryo.
Expression of Erythropoietin Receptor-Like Molecule in Xenopus laevis (xeEPOR) and the Development of Anemia by the Administration of Its Recombinant Soluble Form.
Authors: Aizawa Youichi , Nogawa Nami , Kosaka Nobuyoshi , Maeda Yasutaka , Watanabe Takafumi , Miyazaki Hiroshi , Ochiya Takahiro , Kato Takashi
Published: April 2020

The regulation of hematopoiesis in non-mammalian vertebrates is poorly understood. This is partly because the structures and effects of most hematopoietic regulators have not been identified. As a first step towards studies on the key ingredient of hematopoietic regulation among phyla as well as the diversity of organisms, we have focused on amphibian hematopoiesis. In this study, a cDNA sharing the highest degree of homology with mammalian erythropoietin (EPO) receptors, named xeEPOR tentatively, was cloned from cDNA library of Xenopus immature erythrocytes. The identities of the deduced entire amino acid sequence to human and murine EPO receptors were 24% and 25%, respectively; whereas transmembrane region and motifs of WSXWS and Box1/2 domains were found in the molecule. The northern analysis revealed that two types of xeEPOR RNAs were expressed in normal peripheral blood cells. In addition, by in situ hybridization and immunostaining with monoclonal antibodies raised against the extracellular domain of xeEPOR (soluble xeEPOR), immature basophilic erythrocytes expressing xeEPOR appeared in peripheral blood of phenylhydrazine-treated adult Xenopus. The fulllength xeEPOR cDNA was introduced into murine FDC/P2 cells and the signaling for the cellular proliferation and differentiation was examined in the presence of serum derived from anemic Xenopus as a stimulator. To further understanding the contribution of the xeEPOR gene expression to primitive and definitive hematopoiesis on Xenopus development, whole mount in situ hybridization was performed. As the binding motif of GATA-1, the hematopoietic specific transcription factor, was located at −24 to −15 base upstream of the translation initiation sequence, the correlated expressions of xeEPOR and Xenopus GATA-1 on developing embryo were evaluated with RT-PCR. The xeEPOR RNA was abundantly expressed at Nieuwkoop stage 30 (blood island formation) and thereafter, and temporally followed the expression of GATA-1, suggesting that the functional expression of xeEPOR was upregulated by GATA-1 in Xenopus as reported in studies on mammalian erythropoiesis. To confirm biological functions of the molecule, soluble xeEPOR was administered into adult Xenopus by intracardiac consecutive injections. The peripheral erythrocyte counts were gradually decreased; meanwhile immature erythrocytes were emerged in the circulation, demonstrating that this molecule plays a significant physiological role in erythropoiesis in Xenopus.
Generation of Human Regulatory T Cells Using NOD/SCID/γ CNULL Mice Model.
Authors: Hiramatsu Hidefumi , Fujino Hisanori , Heike Toshio , Ito Mamoru , Nakahata Tatsutoshi
Published: April 2020

We have reported a NOD/SCID/γcnull (NOG) mice model, which enable efficient engraftment of human hematopoietic stem cells and their multi-lineage differentiation including T cells. Using this model, we investigated whether various subpopulations of T cells were generated in this unique murine microenvironment.

Freshly collected cord blood was depleted of phagocytes with Silica ® followed by CD34 positive selection using auto MACS ®. The purity of CD34 positive cells always exceeded 98%. These cells were transplanted into irradiated NOG mice intravenously. About 3 months after the transplantation, human T cells in peripheral blood, bone marrow and spleen were analyzed by flow cytometry. As we have reported previously, more than half of the human cells seen in the spleen were human CD3+ T cells and as many as 30% of them expressed CD4 and CD25 without activation markers such as CD69. To examine if these CD4+ CD25+ cells have regulatory activity, CD4+ CD25− cells were stimulated with anti-human CD3 antibody along with irradiated autologous antigen presenting cells in the presence of limiting dose of CD4+ CD25+ cells. The inhibition of proliferation by CD4+ CD25+ cells was analyzed by 3H-thymidine uptake. CD4+ CD25+ cells successfully suppressed the CD4+ CD25− T cell proliferation and RT-PCR analysis revealed the expression of Foxp3, a marker for regulatory T cells, specifically in the CD4+ CD25+ cell population. These results suggest that regulatory T cells can develop from hematopoietic stem cells in our NOG mice model. As human T cells appear first in the thymus of NOG mice, these regulatory T cells are considered to arise in the murine thymus. Our model provides a new and versatile tool to investigate development and function of human regulatory T cells, which are often difficult to study because of complicated history of infection or genetic differences among individuals.
A Novel Road Map for Blood Lineage Commitment and Development: Identification of a Lympho-Myeloid Stem/Progenitor Cell Lacking Erythro-Megakaryocytic Potential.
Authors: Yang Liping , Aolfsson Jörgen , Månsson Robert , Bryder David , Borge Ole-Johan , Thoren Lina , Sitnicka Ewa , Sasaki Yutaka , Sigvardsson Mikael , Jacobsen Sten Eirik W.
Published: April 2020

The recent identification of common myeloid and lymphoid progenitors (CMPs and CLPs, respectively) lends support to the classical and currently prevailing model for hematopoietic commitment and blood lineage development, suggesting that the first and decisive lineage commitment step of adult hematopoietic stem cells (HSCs) results in an immediate and complete separation of myelopoiesis and lymphopoiesis. Virtually all of multipotent (lympho-myeloid) stem and progenitor cells in adult mouse bone marrow (BM) reside in the small Lin−Sca1+kithi (LSK) compartment. We now present data demonstrating that the LSK compartment in adult BM, can be separated into three phenotypically and functionally distinct HSC subsets, based on differential expression of CD34 and flt3. Long-term HSCs (with extensive self-renewing potential) reside in the LSKCD34−flt3− fraction. The LSKCD34+ short-term HSC compartment consists of LSKCD34+flt3− cells fulfilling all criteria of short-term HSCs, being highly enriched in CFU-S activity, and capable of rapidly reconstituting myelo-erythropoiesis and thereby rescuing myeloablated mice. The short-term LSKCD34+flt3− HSCs give upon transplantation rise to LSKCD34+flt3+ cells, that although sustaining a combined lymphoid (B and T cell) and myeloid (granulocyte and monocyte) developmental potential at the single cell level, loose their ability to adapt megakaryocytic and erythroid fates in vitro as well as in vivo. These evidence for the existence of LSKCD34+flt3+ cells with granulocyte, monocyte, B and T cell, but not megakaryocyte and erythroid development potentials, are not compatible with the first lineage commitment step of HSCs resulting in strict separation of common lymphoid and common myeloid differentiation pathways. Based on the present findings we rather propose an alternative road map for blood lineage development in which LSKCD34+flt3− short-term HSCs genereate megakaryocyte-erythroid progenitors and LSKCD34+flt3+ cells upon asymmetric cell divisions.
Fludarabine Treated CLL Patients Showed an Increased Chemoresistance Ex-Vivo.
Authors: Schinkothe Timo , Hallek Michael J. , Staib Peter
Published: April 2020

Fludarabine is the most successful drug in patients with relapsed, progradient or refractory chronic lymphocytic leukemia (CLL). But also the use of fludarabine in primary therapy as an alternative to chlorambucil is in trial. Fludarabine induces higher response rates, but showed no improvement in overall survival. The reason for this clinical observation is unclear. In the present study we wanted to answer the question, if the induction of secondary resistance might be the cause for this contradiction.

METHODS. Peripheral blood samples from 42 patients with B-CLL were tested in the in-vitro chemosensitivity-test DiSC-assay with 11 drugs. 10 patients had received no therapy before the time of carrying out the In vitro test. 23 patients were pre-treated with prednisone, 8 patient with fludarabine and 5 patients with vincristine. Most of the patients received more then one drug before testing. The chemosensitivity were determined using the LC90.

RESULTS. The patients were divided into those who were pre-treated vs. not pre-treated with the fludarabine, prednisone or vincristine. The influences of pre-treatment to the sensitivity were analysed using the Mann-Whitney-U test for two independent groups. With fludarabine pre-treated patients had an significantly increased chemoresistance against fludarabine (2.4-fold; p=.004). The increase was also significant for bendamustin (4.4-fold), 2-CdA (3.4-fold), Gemcitabine (2.7-fold) and daunorubicin (1.5-fold). There was no significant influence observed onto the other drugs. Pre-treatment with vincristine or prednisone had no impact onto the chemosensitivity of any tested drug.

INTERPRETATION AND CONCLUSIONS. The results of this study demonstrate the capability of fludarabine to increase the chemoresistance against various drugs in CLL. The use of fludarabine in primary therapy should be discussable. The fact that fludarabine induces higher response rates, without an improvement in overall survival, might be understandable if the fludarabine treatment induces a negative long-term effect. The widely increase of the chemoresistance is such a negative long-term effect. Based on the presented data, we postulate that fludarabine induces a so called secondary resistance by selecting leukemic cells with a lower chemosensitivity under treatment.
Inhibition of Protein Kinase C (PKC) delta with Rottlerin Induces Apoptosis in B-CLL Cells, Augments Chemotherapeutic Cytotoxicity and Inhibits Antiapoptotic Survival Signals.
Authors: Ringshausen Ingo , Weick Kathrin , Oelsner Madlene , Peschel Christian , Decker Thomas
Published: April 2020

Purpose: We have previously shown that PKC delta is constitutively activated in B-CLL cells. In the present study, we have analyzed the mechanism of apotosis induction as well as the effets of PKC delta in the presence of survival signal and chemotherapeutic agents.

Methods: The mitochondrial membrane potential, presence of active caspase 3, conformational status of Bax and apoptosis assays (Annexin V stain, Tunel assay) were performed using flow cytometric analysis. Expression of BCL-2 family proteins and XIAP as well as processing of caspase 8 and 9 was revealed in western blot experiments.

Results: Rottlerin at 5μM (which is not toxic for normal lymphocytes) induced apoptosis in a large amount of B-CLL samples. Rottlerin was equally effective in zap70 positive- and negative samples. Rottlerin induced apoptosis was in part caspase dependent and involved processing of caspase 3, 8 and 9. Expression of antiapoptotic proteins MCL-1 and XIAP was reduced in Rottlerin treated cells and BAX expression increased and Bax conformation changed to its proapoptotic form.IN contrast, expression of bcl-2 was not changed.

B-CL cells are likely to receive survival signals from the microenvironment and might be rescued from cell death induced by chemotherapeutic agents. Rottlerin was very effective in inhibiting survival signals like IL-4 or stromal cell contact. In addition, treatment with Rottlerin enhanced the cytotoxicity of the chemotherapeutic agents Fludarabine, Vncristine and Daunorubicine.

Conclusion: Inhibition of PKC delta might be a powerful treatment option for B-CLL given its potential to induce apoptosis, inhibit antiapoptotic survival signals and augment the cytotoxicity of chemotherapeutic agents.
Chronic Lymphocytic Leukemia Cells Are Dependent on Autocrine VEGF and α4β1 Integrin for Chemokine-Induced Motility.
Authors: Till Kathleen J. , Spiller David G. , Harris Robert J. , Chen Haijuan , Zuzel Mirko , Cawley John C.
Published: April 2020

VEGF is a multifunctional cytokine involved in tumourigenesis. In chronic lymphocytic leukaemia (CLL), it is known that the malignant cells secrete VEGF and possess VEGF receptors. This suggests that an autocrine loop might be important in the pathogenesis of CLL. Here we show that, in patients with lymphadenopathy, autocrine VEGF and α4β1-integrin are involved in the chemokine-dependent motility of CLL cells on and through endothelium - processes important for the invasion of lymphoreticular tissues. In contrast, normal lymphocytes were not dependent on autocrine VEGF or α4β1 for either type of cell movement. Moreover, in contrast to normal B lymphocytes, CLL cells failed to cluster and activate αLβ2 in response to chemokines, unless VEGF receptor(s) and α4β1 were also engaged by their respective ligands. This is the first demonstration that autocrine VEGF is involved in CLL-cell motility and that the αLβ2 on the malignant cells is functionally altered in the disease. Taken together, the results suggest that VEGF and α4 blockade may be therapeutically useful in CLL patients with tissue disease.
The Incidence of Chronic Lymphocytic Leukemia in the General Population.
Authors: Frezzato Maurizio , Tosetto Alberto , Giaretta Ilaria , Rodeghiero Francesco
Published: April 2020

Background. Chronic Lymphocytic Leukemia is the most frequent leukemia in the Western countries (Rozman, 1995) but its incidence is not well established. Epidemiological data are based on reports from tumour registries, with an estimated incidence lower than 3-cases/100000 pts-year. Tumour registries can however underestimate the incidence of CLL as highlighted by the 37% higher incidence reported in a selected population of Veterans (Zent, 2000). We aimed to assessing the incidence of CLL in a population-based cohort from an epidemiological survey on familial thrombosis, (Tosetto, 2003) which was followed for at least 8 years.

Material and Methods. 15109 healthy people from 18 to 65 years of age in the Vicenza township were enrolled in the VITA Project (8017 females and 7092 males, with a median age of 43 years). A clinical history was collected and blood and DNA samples were withdrawn and stored at the time of enrolment. The study begun in 1993 and ended in 1996. Patients with a diagnosis of CLL (ICD-9 code 204) were traced using data from the Veneto Region Health Service database, which records all hospital admission for residents in the enrolment area. Moreover, the database encompasses data of all the subjects visited in the Vicenza Hematology Department, the unique hematological facility of the area, even if not subsequently admitted to the hospital. Diagnosis of CLL was validated if based on morphological and immunophenotypic data according to currently accepted criteria (Cheson, 1988; IWCC, 1989; Rozman, 1995).

Results. Six out 15109 people were diagnosed with a CLL (4 males). The median follow-up for the entire cohort was 8.2 years and the total time of exposure was 121000 pts-year. Incidence of CLL is estimated to be 4.9 cases/100000 pts-year (CI: 1.8–10.7). Diagnosis of CLL was done from 36 to 97 months after the enrolment. Mean age at diagnosis was 66 years (61 – 70). At the time of enrolment in the study, all of the patients had a normal WBC. Three persons presented an immunoglobulin VH gene rearrangement, 32, 54 and 57 months before diagnosis. All of the patients were asymptomatic at diagnosis. The median follow-up of all the subjects was 28 months (7 – 57 months). Only one of them became symptomatic and needed a treatment, 40 months after the diagnosis.

Conclusions. According to our data, the incidence of CLL appears to be higher than assessed so far. In our opinion it’s noteworthy that the estimation is based on data of subjects evaluated in a clinical setting and not from tumour or disease registries. In fact it could be still underestimated comparing the young median age of our cohort with that usually reported at diagnosis (Rozman, 1995). Moreover, the comparison of our data with those available could suggest that only a quote of the cases might be usually diagnosed. Finally, the presence of the Ig VH gene rearrangement at the time of the enrolment suggests that the disease may be present a very long time before any clinical evidence.
Final Results of the Thai-NHLBI Epidemiological Study of Aplastic Anemia.
Authors: Issaragrisil Surapol , Kaufman David W. , Anderson Theresa E. , Chansung Kanchana , Leaverton Paul , Shapiro Samuel , Young Neal S.
Published: April 2020

Aplastic anemia, rare in the West, is a relatively more frequent hematologic diagnosis in Asia. A systematic epidemiological study of aplastic anemia was conducted in Thailand from 1989–2002 in order to determine both the incidence and environmental risk factors. We employed the methodology established by the International Agranulocytosis and Aplastic Anemia Study, conducted in Europe and Israel in the 1980s. In the first phase of the current study, we documented an annual incidence of 3.9 per million for the Bangkok metropolitan area and 5 per million in the northeast region of Khonkaen. Aplastic anemia therefore occurs at a rate at least 2–3 fold more frequent in Thailand than in Europe and Israel. We now report final results of the case-control portion of the study, in which 541 patients and 2,261 hospital controls were enrolled--the largest epidemiologic study of aplastic anemia to date. Environmental exposures were determined with a detailed personal interview, with emphasis on both known risk factors for aplastic anemia and novel factors that might provide clues to disease causation. Among previously suspected factors, we observed significantly elevated relative risk estimates for benzene (3.5) and other solvents (mostly unspecified thinners; 2.0), and among medical drugs for sulfonamides (5.6), thiazides (3.8), and mebendazole (3.0). Chloramphenicol use was infrequent among cases and controls, and no significant association was observed. A number of agricultural pesticides were implicated in Khonkaen: there were significant associations with organophosphates (2.1), DDT (6.7), and carbamates (7.4), and an association of borderline significance with paraquat (2.3). Among novel factors, we found significant risks for farmers and their family members who had been exposed to ducks and geese (3.7) and a borderline association with the use of animal fertilizer (2.1). There was a significant association confined to Khonkaen with drinking other than bottled and distilled water (2.8). Nonmedical needle exposure through tattooing, body piercing, or acupuncture was infrequent but associated in Bangkok and Khonkaen combined (3.8). Only two cases consistent with a diagnosis of post-hepatitis aplastic anemia were observed. Our results confirm the well known link between aplastic anemia and benzene, other solvents, and some medical drugs, but only a small proportion of cases were exposed to these agents. A variety of pesticides were implicated and these could account for up to 15% of cases in Khonkaen. Most striking was the large etiologic fraction of aplastic anemia in that region accounted for by animal exposures and especially drinking of water from sources such as wells, rural taps, and rainwater. These findings are consistent with an infectious etiology for many cases of aplastic anemia in rural Thailand.
PIG-A Mutations in Normal Hematopoiesis.
Authors: Hu Rong , Mukhina Galina , Piantadosi Steven , Jones Richard J. , Brodsky Robert A.
Published: April 2020

Introduction: Paroxysmal nocturnal hemoglobinuria (PNH) is caused by somatic mutations of the X-linked gene, PIG-A, in hematopoietic stem cells (HSCs). The product of this gene is necessary for the assembly of glycosylphosphatidylinositol (GPI) anchors. Consequently, PNH cells lack the expression of GPI-anchored proteins on their cell surface. PIG-A mutations have been found in granulocytes and T lymphocytes from most normal individuals. Although the significance of these mutations is unclear, it suggests that they are important in the pathogenesis of PNH.

Methods: We isolated CD34+ progenitors from 4 PNH patients, 18 healthy donors, and 9 non-PNH patients undergoing peripheral blood stem cell tranplantation. The frequency of PIG-A mutant progenitors was determined by assaying for colony forming cells (CFC) in methylcellulose containing toxic doses of aerolysin. Aerolysin is a pore-forming toxin that uses the GPI anchor as it receptor; hence, PNH cells are unique in their resistance to aerolysin. DNA was extracted from individual day 14 aerolysin resistant CFC and the PIG-A gene was sequenced to determine clonality. We performed a Poisson distribution of the mutational frequency to determine the probability that the PIG-A mutation in controls arose from HSC.

Results: In PNH patients, 67% of the CFC were aerolysin resistant. The frequency of aerolysin resistant CFC was 14.7 ± 4.0 x 10−6 in the bone marrow of healthy donors and was 57.0 ± 6.7 x 10−6 from mobilized peripheral blood. Aerolysin resistant CFC from PNH patients exhibited clonalPIG-A mutations, and thus, arose from HSC. In contrast, PIG-A mutations in the CFC from controls were polyclonal (up to 15 different mutations from one individual). Recent evidence suggests that humans and other large mammals possess only 10,000 primitive HSC, and that only 1000 of these cells are thought to contribute to hematopoiesis at any one time. Poisson statistics show that only 5% of normals would be expected to harbor a PIG-A mutation in 1000 HSC, and < 1 x 10−9 persons would harbor 10 or more different PIG-A mutations even if all 10,000 hematopoietic stem cells were contributing to hematopoiesis. Thus, the high frequency of PIG-A mutations in controls, coupled with their polyclonality, suggests that they do not arise at the level of HSC; rather, PIG-A mutations in normals appear to arise as a consequence of hematopoietic differentiation, between the level of an HSC and a CFC.

Conclusion: Our data confirm the findings that PIG-A mutations are relatively common in normal hematopoiesis. Although we cannot rule out that a rare PIG-A mutant blood cell in normals does in fact arise from a mutant HSC, our data suggest that most of the mutations occur with differentiation. Genetic fidelity can be lost with differentiation without consequence, as mutations in differentiated cells would not be propagated. These data also call into question the relevance of PIG-A mutations in normals to the pathogenesis of PNH.
Nuclear Localization of Fanconi Anemia Protein FANCA Is Regulated by Hsc70/Hsp90 Chaperone Machinery.
Authors: Oda Tsukasa , Miyaso Hidenobu , Yamashita Takayuki
Published: April 2020

Fanconi anemia (FA) is a genetic disorder characterized by bone marrow failure, cancer susceptibility and cellular hypersensitivity to DNA crosslinkers such as mitomycin C (MMC). Current evidence indicates that formation of a nuclear multiprotein complex (core complex) including six FA proteins FANCA/C/E/F/G/L is essential for FANCL/PHF9 ubiquitin ligase-mediated activation of FANCD2 into a monoubiquinated form, which participates in BRCA1 and FANCD1/BRCA2-mediated DNA repair (the FA/BRCA pathway). Subcellular distribution of FANCA plays a crucial role in the regulation of the FA/BRCA pathway. However, the underlying molecular mechanisms are not fully understood. To address this issue, we tried to identify FANCA-associated proteins. To this end, Flag-FANCA ectopically expressed in HeLa cells was immunopurified from the cytoplasmic fraction, using anti-Flag antibody-conjugated sepharose beads. Analysis of the immune complex on SDS polyacrylamide gel electrophoresis revealed that several proteins of Mr. 60–70 kD specifically associated with Flag-FANCA. These proteins were identified as FANCG and Hsc (heat shock cognate protein) 70 by LC-MS/MS. Immunoblot analysis showed that FANCA associated with Hsp90 as well as Hsc70. Hsc70 is an ATP-dependent molecular chaperone highly homologous to Hsp70 and often cooperates with Hsp90 to form a chaperone machinery involved in the regulation of diverse protein functions. Patient-derived FANCA mutants failed to bind FANCC but associated with larger amounts of Hsc70 than wt-FANCA, indicating that the interaction between FANCA and Hsc70 is not mediated by FANCC, as suggested by previous observations of the interaction of FANCC with Hsp70. To study the role of Hsc70 and Hsp90 in the regulation of FANCA, we examined effects of a dominant-negative (dn) form of Hsc70 with inactivated ATPase activity, and a specific inhibitor of Hsp90, 17-AAG (a geldanamycin analog). Overexpression of dn-Hsc70 inhibited nuclear localization of FANCA and inhibited its core complex formation, whereas wt-Hsc70 did not. 17-AAG induced cytoplasmic distribution and proteosomal degradation of FANCA and suppressed FANCD2 mono-ubiquitination. Taken together, these results suggest that Hsc70/Hsp90 chaperone machinery interacts with FANCA and regulates its subcellular distribution and stability, thereby controlling activation of the FA/BRCA pathway.