bluuebunny/crossref_metadata_2025 huggingface dataset:
The Cellular Labile Iron Pool and Intracellular Ferritin in K562 Cells
Authors: Konijn Abraham M. , Glickstein Hava , Vaisman Boris , Meyron-Holtz Esther G. , Slotki Itzchak N. , Cabantchik Z. Ioav
Published: October 2019

The labile iron pool (LIP) harbors the metabolically active and regulatory forms of cellular iron. We assessed the role of intracellular ferritin in the maintenance of intracellular LIP levels. Treating K562 cells with the permeant chelator isonicotinoyl salicylaldehyde hydrazone reduced the LIP from 0.8 to 0.2 μmol/L, as monitored by the metalo-sensing probe calcein. When cells were reincubated in serum-free and chelator-free medium, the LIP partially recovered in a complex pattern. The first component of the LIP to reappear was relatively small and occurred within 1 hour, whereas the second was larger and relatively slow to occur, paralleling the decline in intracellular ferritin level (t½= 8 hours). Protease inhibitors such as leupeptin suppressed both the changes in ferritin levels and cellular LIP recovery after chelation. The changes in the LIP were also inversely reflected in the activity of iron regulatory protein (IRP). The 2 ferritin subunits, H and L, behaved qualitatively similarly in response to long-term treatments with the iron chelator deferoxamine, although L-ferritin declined more rapidly, resulting in a 4-fold higher H/L-ferritin ratio. The decline in L-ferritin, but not H-ferritin, was partially attenuated by the lysosomotrophic agent, chloroquine; on the other hand, antiproteases inhibited the degradation of both subunits to the same extent. These findings indicate that, after acute LIP depletion with fast-acting chelators, iron can be mobilized into the LIP from intracellular sources. The underlying mechanisms can be kinetically analyzed into components associated with fast release from accessible cellular sources and slow release from cytosolic ferritin via proteolysis. Because these iron forms are known to be redox-active, our studies are important for understanding the biological effects of cellular iron chelation.
Posttranslational Processing of the Thrombopoietin Receptor Is Impaired in Polycythemia Vera
Authors: Moliterno Alison R. , Spivak Jerry L.
Published: October 2019

Recently, we demonstrated a marked reduction in the expression of the thrombopoietin receptor, Mpl, in polycythemia vera (PV) platelets and megakaryocytes using an antiserum against the Mpl extracellular domain. To further examine this abnormality, we raised an antibody to the Mpl C-terminus. Immunologic analysis of PV platelets with this antiserum confirmed the reduction in Mpl expression. However, the C-terminal antiserum detected 2 forms of Mpl in PV platelets in contrast to normal platelets, in which a single form of Mpl was detected by both the extracellular domain and C-terminal antisera. Two-dimensional gel electrophoresis studies with isoelectric focusing in the first dimension identified normal platelet Mpl as an 85 to 92 kD protein with an isoelectric point (pI) of 5.5. PV platelets contained an additional 80 to 82 kD Mpl Mpl isoform with a pI of 6.5. Analysis of Mpl expressed by the human megakaryocytic cell line, Dami, showed 2 isoforms similar to those found in PV platelets suggesting a precursor-product relationship. Digestion of Dami cell and normal platelet lysates with neuraminidase converted the more acidic Mpl isoform to the more basic one, indicating that the 2 isoforms differed with respect to posttranslational glycosylation. Futhermore, in contrast to normal platelet Mpl, PV platelet Mpl was susceptible to endoglycosidase H digestion, indicating defective Mpl processing by PV megakaryocytes. The glycosylation defect was specific for Mpl, as 2 other platelet membrane glycoproteins, glycoprotein IIb and multimerin, were processed normally. Importantly, the extent of the PV platelet Mpl glycosylation defect correlated with disease duration and extramedullary hematopoiesis.
Unique Differentiation Programs of Human Fetal Liver Stem Cells Shown Both In Vitro and In Vivo in NOD/SCID Mice
Authors: Nicolini Franck E. , Holyoake Tessa L. , Cashman Johanne D. , Chu Pat P.Y. , Lambie Karen , Eaves Connie J.
Published: October 2019

Comparative measurements of different types of hematopoietic progenitors present in human fetal liver, cord blood, and adult marrow showed a large (up to 250-fold), stage-specific, but lineage-unrestricted, amplification of the colony-forming cell (CFC) compartment in the fetal liver, with a higher ratio of all types of CFC to long-term culture-initiating cells (LTC-IC) and a lower ratio of total (mature) cells to CFC. Human fetal liver LTC-IC were also found to produce more CFC in LTC than cord blood or adult marrow LTC-IC, and more of the fetal liver LTC-IC–derived CFC were erythroid. Human fetal liver cells regenerated human multilineage hematopoiesis in NOD/SCID mice with the same kinetics as human cord blood and adult marrow cells, but sustained a high level of terminal erythropoiesis not seen in adult marrow-engrafted mice unless exogenous human erythropoietin (Epo) was injected. This may be due to a demonstrated 10-fold lower activity of murine versus human Epo on human cells, sufficient to distinguish between a differential Epo sensitivity of fetal and adult erythroid precursors. Examination of human LTC-IC, CFC, and erythroblasts generated either in NOD/SCID mice and/or in LTC showed the types of cells and hemoglobins produced also to reflect their ontological origin, regardless of the environment in which the erythroid precursors were generated. We suggest that ontogeny may affect the behavior of cells at many stages of hematopoietic cell differentiation through key changes in shared signaling pathways.
Circulating Activated Platelets Assist THP-1 Monocytoid/Endothelial Cell Interaction Under Shear Stress
Authors: Theilmeier Gregor , Lenaerts Tim , Remacle Claude , Collen Désiré , Vermylen Jos , Hoylaerts Marc F.
Published: October 2019

Circulating complexes of leukocytes and activated platelets are markers for atherosclerosis, but their interaction with the arterial endothelial lining has not been studied. Therefore, the effect of activated platelets on rolling and adhesion of labeled human THP-1 monocytoid cells to human umbilical vein endothelial cell (HUVEC) monolayers was studied by epifluorescence microscopy in a parallel plate flow chamber. In the absence of activated platelets, THP-1 rolling on resting HUVEC was negligible at shear rates greater than 300 s−1. Activation of HUVEC with 100 nmol/L phorbol myristate acetate (PMA) increased THP-1 cell adhesion at shear rates less than 400 s−1. Therefore, a shear rate of 400 s−1 was identified as a threshold for THP-1 adhesion. THP-1 rolling on activated HUVEC was reduced by 64% after L-selectin inhibition but was not affected by P-selectin inhibition. The addition of 1 to 50 thrombin receptor-activating peptide (TRAP)-activated platelets per THP-1 cell enhanced interactions between THP-1 cells and HUVEC, resulting in a steep bell-shaped dose-response curve, with a peak of 10 ± 3 rolling cells/50 seconds at 3 platelets per THP-1 cell (P < .01v control) with a concomitant 2- to 3-fold increase of firmly adhering cells (P < .01 v control). In reconstituted blood, low numbers of activated platelets had the same effect on THP-1 rolling and adhesion. P-selectin inhibition reduced platelet/THP-1 cell interaction in suspension and deposition of the complexes on the endothelial monolayer. Inhibition of both P- and L-selectin reduced THP-1/HUVEC interactions to 14% (P < .01, n = 4). Sialidase digestion and removal of terminal sialic acid residues from HUVEC or THP-1 cells but not from platelets abolished the platelet mediated augmentation of THP-1 cell adhesion. Thus, THP-1 rolling on HUVEC is shear-dependent and largely mediated by L-selectin. P-selectin expressed on activated platelets increases monocytoid cell adhesion to endothelial cells at shear rates found in coronary arteries through interactions with both endothelial and monocytoid cells and may facilitate macrophage accumulation in the vessel wall.
ABO Blood Group Antigens on Human Plasma von Willebrand Factor After ABO-Mismatched Bone Marrow Transplantation
Authors: Matsui Taei , Shimoyama Taketo , Matsumoto Masanori , Fujimura Yoshihiro , Takemoto Yoshinobu , Sako Masahiro , Hamako Jiharu , Titani Koiti
Published: October 2019

von Willebrand factor (vWF) is synthesized exclusively by endothelial cells and megakaryocytes, and stored in the intracellular granules or constitutively secreted into plasma. ABO blood group antigens are covalently associated with asparagine-linked sugar chains of plasma vWF. The effect of ABO-mismatched bone marrow transplantation (BMT) or blood stem cell transplantation (BSCT) on the expression of ABO blood group antigens on the vWF was examined to obtain information on the origin of these antigens. In ABO-mismatched (HLA-matched) groups, 8 cases of BMT and 4 cases of BSCT were examined. In all cases, the ABO blood groups on red blood cells were gradually converted to the donor’s type within 80 to 90 days after the transplantation. The blood group antigens on the vWF were consistent with the recipient’s blood group for the period monitored by enzyme-linked immunosorbent assay (ELISA). When vWF was isolated from normal platelets and examined for the blood group antigens using ELISA or immunoblotting, it showed few antigens. However, vWF extracted from veins expressed blood group antigens. These findings indicate that platelet (megakaryocyte)-derived vWF does not contain blood group antigens and that these antigens may be specifically associated with vWF synthesized in endothelial cells and secreted into plasma. Furthermore, it is possible that the persistence of the recipient’s blood group antigens on plasma glycoproteins such as vWF, independent of the donor-derived erythrocytes, after ABO-mismatched stem cell transplantation, may influence the immunological system in the production of anti-blood group antibodies resulting in the establishment of immunological tolerance in the recipient plasma.
Distinct signals control the hematopoiesis of lymphoid-related dendritic cells
Authors: Galy Anne , Christopherson Indu , Ferlazzo Guido , Liu Guo , Spits Hergen , Georgopoulos Katia
Published: October 2019

The molecular and cellular requirements for the development of different populations of human dendritic cells (DC) were studied. Conditions were defined that support DC production from lymphoid progenitors but that fail to induce DC formation from peripheral monocytes. The production of these lymphoid-related DC was severely blocked when hematopoietic progenitors overexpressed Ik7, a mutant dominant-negative Ikaros protein. In contrast, Ik7 did not block the formation of DC in conditions supporting the development of monocyte-derived DC. Furthermore, Ik7 did not block the formation of monocyte/macrophages and enhanced granulopoiesis. One of the molecular mechanisms mediated by Ik7 appears to be down-regulation of the flt3-receptor mRNA. Thus, distinct signals control the formation of DC demonstrating that some aspects of DC diversity are determined in part by distinct molecular cues at the hematopoietic level. (Blood. 2000;95:128-137)
Protein S secretion differences of missense mutants account for phenotypic heterogeneity
Authors: Espinosa-Parrilla Yolanda , Yamazaki Tomio , Sala Nùria , Dahlbäck Björn , de Frutos Pablo Garcı́a
Published: October 2019

To elucidate the molecular background for the heterogeneity in protein S plasma concentrations observed in protein S deficient individuals, the in vitro synthesis of recombinant protein S missense mutants was investigated. Six different naturally occurring mutations identified in the protein S gene (PROS1) of thrombosis patients were reproduced in protein S cDNA by site directed mutagenesis. Two mutants, G441C and Y444C (group A), were associated with low total plasma concentration of protein S. Modestly low protein S was found in families with R520G and P626L (group B) mutants. T57S and I518M (group C), which was associated with marginally low protein S, did not segregate with protein S deficiency in the respective families, raising doubts as to whether they were causative mutations or rare neutral variants. The 6 protein S mutants were transiently expressed in COS 1 cells. The Y444C mutant showed the lowest level of secretion (2.5%) followed by the G441C mutant (40%). Group B demonstrated around 50% reduction in secretion, whereas group C mutants showed normal secretion. Pulse-chase experiments demonstrated impaired protein S processing with intracellular degradation and decreased secretion into the culture media of group A and B mutants. Interestingly, there was a good correlation between in vitro secretion and the concentration of free protein S in the plasma of heterozygous carriers. These results demonstrate impaired protein S secretion to be an important mechanism underlying hereditary protein S deficiency and that variations in protein secretion is a major determinant of the phenotypic heterogeneity observed in protein S deficiency. (Blood. 2000;95:173-179)
A naturally occurring mutation near the amino terminus of αIIb defines a new region involved in ligand binding to αIIbβ3
Authors: Basani Ramesh B. , French Deborah L. , Vilaire Gaston , Brown Deborah L. , Chen Fangping , Coller Barry S. , Derrick Jerry M. , Gartner T. Kent , Bennett Joel S. , Poncz Mortimer
Published: October 2019

Decreased expression of functional IIbβ3 complexes on the platelet surface produces Glanzmann thrombasthenia. We have identified mutations of IIbP145 in 3 ethnically distinct families affected by Glanzmann thrombasthenia. Affected Mennonite and Dutch patients were homozygous and doubly heterozygous, respectively, for a P145A substitution, whereas a Chinese patient was doubly heterozygous for a P145L substitution. The mutations affect expression levels of surface IIbβ3 receptors on their platelets, which was confirmed by co-transfection of IIbP145A and β3 cDNA constructs in COS-1 cells. Each mutation also impaired the ability of IIbβ3 on affected platelets to interact with ligands. Moreover, when IIbP145A and β3 were stably coexpressed in Chinese hamster ovary cells, IIbβ3 was readily detected on the cell surface, but the cells were unable to adhere to immobilized fibrinogen or to bind soluble fluorescein isothiocyanate–fibrinogen after IIbβ3 activation by the activating monoclonal antibody PT25-2. Nonetheless, incubating affected platelets with the peptide LSARLAF, which binds to IIb, induced PF4 secretion, indicating that the mutant IIbβ3 retained the ability to mediate outside-in signaling. These studies indicate that mutations involving IIbP145 impair surface expression of IIbβ3 and that the IIbP145A mutation abrogates ligand binding to the activated integrin. A comparative analysis of other IIb mutations with a similar phenotype suggests that these mutations may cluster into a single region on the surface of the IIb and may define a domain influencing ligand binding. (Blood. 2000;95:180188)
Fibrinogen is a component of a novel lipoprotein particle: Factor H–related protein (FHRP)–associated lipoprotein particle (FALP)
Authors: Park C. Thomas , Wright Samuel D.
Published: October 2019

We have previously described a novel lipoprotein particle consisting of phospholipids, apolipoprotein A-I (apoAI), lipopolysaccharide binding protein (LBP) and Factor H-related proteins (FHRP), and we termed these particles FALP (FHRP-associated lipoprotein particles). Highly purified preparations of FALP contain variable amounts of an unidentified polypeptide triplet of Mr ∼85 000 (tp85). Here we report that tp85 represents fragment D of fibrinogen, as confirmed by N-terminal amino acid sequencing and Western blot analysis with an antifibrinogen antibody. The physical association of fibrinogen with other components of FALP in plasma was further confirmed by sandwich ELISA by using monoclonal antibodies against apoAI, FHRP or LBP to capture the particles and polyclonal antifibrinogen as the detecting antibody. Furthermore, affinity chromatography with anti-FHRP-1–specific IgG showed that fibrinogen is co-immunodepleted with FALP and approximately 17% of total plasma fibrinogen are bound to FALP. LBP is a lipid transfer protein that moves lipopolysaccharide (LPS) to a binding site on CD14 or high-density lipoprotein (HDL). To determine whether fibrinogen affects the lipid transfer activity of LBP on FALP, this activity was measured in FALP prepared with and without fibrinogen. Neither activity of LBP was affected by fibrinogen. The abundance of FALP suggests, instead, an effect of FALP on the function or clearance of fibrinogen or fragment D. (Blood. 2000;95:198-204)
Nitric oxide-producing CD11b+Ly-6G(Gr-1)+CD31(ER-MP12)+cells in the spleen of cyclophosphamide–treated mice: implications for T-cell responses in immunosuppressed mice
Authors: Angulo Iñigo , de las Heras Federico Gómez , Garcı́a-Bustos José F. , Gargallo Domingo , Muñoz-Fernández M. Angeles , Fresno Manuel
Published: October 2019

During recovery from intensive chemotherapy with cyclophosphamide (CTX), mice suffer a severe but transitory impairment in spleen cell proliferation to T-cell mitogens (Con A or anti-CD3 plus IL-2). Although CTX treatment reduced spleen T-cell cellularity, this cannot fully account for T-cell unresponsiveness. The results showed that CTX induces the colonization of spleen by an immature myeloid CD11b+Ly-6G+CD31+ population. Its presence closely correlated with the maximum inhibition of T-cell proliferation. Moreover, this suppressive activity was dependent on nitric oxide (NO) production in cultures since (1) higher amounts of nitric oxide and inducible nitric oxide synthase (iNOS) mRNA were produced in CTX spleen cells (CTX-SC) than in control splenocyte cultures and (2) NOS inhibitors greatly improved the proliferation of T lymphocytes. Nitric oxide production and suppressive activity were also dependent on endogenous interferon-γ (IFN-γ) production since anti–IFN-γ abrogated both effects. Finally, iNOS protein expression was restricted to a heterogeneous population of CD31+cells in which CD11b+Ly-6G+ cells were required to suppress T-cell proliferation. These results indicated that CTX might also cause immunosuppression by a mechanism involving the presence of immature myeloid cells with suppressor activity. This may have implications in clinical praxis since inappropriate immunotherapies in patients treated with intensive chemotherapy could lead to deleterious T-cell responses. (Blood. 2000;95:212-220)
A splice variant of human ephrin-A4 encodes a soluble molecule that is secreted by activated human B lymphocytes
Authors: Aasheim Hans-Christian , Munthe Else , Funderud Steinar , Smeland Erlend B. , Beiske Klaus , Logtenberg Ton
Published: October 2019

Ephrin-A4 is a ligand for the erythropoietin-producing hepatocellular (Eph) receptor family of tyrosine kinases. We have identified a secreted form of ephrin-A4, denoted ephrin-A4 (s), which is encoded by an alternatively spliced mRNA and is produced by in vivo activated B cells in tonsils. Blood B cells secrete ephrin-A4 (s) upon stimulation via the B-cell antigen receptor. A subpopulation of tonsil cells in the crypts with a dendritic cell phenotype was shown to express EphA2, an Eph receptor tyrosine kinase that was found to be capable of binding an ephrin-A4 immunoglobulin chimeric protein. We conclude that ephrin-A4 (s) may play a role in the interaction between activated B lymphocytes and dendritic cells in human tonsils. (Blood. 2000;95:221-230)
Inhibition of eosinophilic inflammation in allergen-challenged, IL-1 receptor type 1–deficient mice is associated with reduced eosinophil rolling and adhesion on vascular endothelium
Authors: Broide David H. , Campbell Keith , Gifford Tim , Sriramarao P.
Published: October 2019

To determine the relative in vivo importance of IL-1 release after allergen challenge to the subsequent endothelial adhesion and recruitment of eosinophils, the authors used ovalbumin sensitization and inhalation challenge to induce airway eosinophilia in IL-1 receptor type 1-deficient and control wild-type mice. Bronchoalveolar lavage (BAL) eosinophil recruitment in IL-1 receptor type 1-deficient mice challenged with ovalbumin (24.3% ± 6.3% BAL eosinophils) was significantly reduced compared with wild-type mice (63.7% ± 2.5% BAL eosinophils). To determine whether the inhibition of eosinophil adhesion to vascular endothelium contributed to the inhibition of eosinophil recruitment in IL-1 receptor type 1-deficient mice, the authors used intravital microscopy to visualize the rolling and firm adhesion of fluorescence-labeled mouse eosinophils in the microvasculature of the allergen-challenged mouse mesentery. Eosinophil rolling, eosinophil firm adhesion to endothelium, and transmigration across endothelium (peritoneal eosinophils) were significantly inhibited in allergen-challenged IL-1 receptor type 1-deficient mice compared with wild-type mice. Overall, these studies demonstrate that cytokines such as IL-1, released after allergen challenge, are important in the induction of endothelial cell adhesiveness, a prerequisite for the recruitment of circulating eosinophils. (Blood. 2000;95:263-269)
HLA class I-restricted lysis of leukemia cells by a CD8+ cytotoxic T-lymphocyte clone specific for WT1 peptide
Authors: Ohminami Hideki , Yasukawa Masaki , Fujita Shigeru
Published: October 2019

The Wilms tumor (WT1) gene has been reported to be preferentially expressed in acute leukemia cells, regardless of leukemia subtype and chronic myelogenous leukemia cells in blast crisis, but not in normal cells. This finding suggests strongly that WT1 protein is a potential target of immunotherapy for human leukemia. In this study, we established a CD8+ cytotoxic T-lymphocyte (CTL) clone directed against a WT1-derived peptide and examined its immunologic actions on leukemia cells. A CD8+ CTL clone, designated TAK-1, which lysed autologous cells loaded with a WT1-derived 9-mer peptide consisting of the HLA-A24 (HLA-A*2402)-binding motifs was established by stimulating CD8+ T lymphocytes from a healthy individual repeatedly with WT1 peptide-pulsed autologous dendritic cells. TAK-1 was cytotoxic to HLA-A24–positive leukemia cells expressing WT1, but not to HLA-A24–positive lymphoma cells that did not express WT1, HLA-A24–negative leukemia cells, or HLA-A24–positive normal cells. Treating leukemia cells with an antisense oligonucleotide complementary to the WT1 gene resulted in reduced TAK-1-mediated cytotoxicity, suggesting that target antigen of TAK-1 on leukemia cells is the naturally processed WT1 peptide in the context of HLA-A24. TAK-1 did not inhibit colony formation by normal bone marrow cells of HLA-A24–positive individuals. Because WT1 is overexpressed ubiquitously in various types of leukemia cells, but not in normal cells, immunotherapy using WT1 peptide-specific CTL clones should be an efficacious treatment for human leukemia. (Blood. 2000;95:286-293)
Altered control of self-reactive IgG by autologous IgM in patients with warm autoimmune hemolytic anemia
Authors: Stahl Dorothea , Lacroix-Desmazes Sébastien , Heudes Didier , Mouthon Luc , Kaveri Srini V. , Kazatchkine Michel D.
Published: October 2019

Warm autoimmune hemolytic anemia (WAIHA) is characterized by an accelerated clearance of red blood cells (RBCs) associated with the presence of anti-RBC immunoglobulin (Ig)G autoantibodies. In the present study, we analyzed the self-reactive IgG and IgM antibody repertoires of patients with WAIHA using a technique of quantitative immunoblotting on a panel of whole tissue extracts as sources of self-antigens. Data were compared by means of multiparametric statistical analysis. We demonstrate that self-reactive antibody repertoires of IgG purified from plasma and of IgG purified from RBC eluates do not differ between healthy donors and patients with WAIHA, whereas autoreactive repertoires of IgM from patients exhibit broadly altered patterns of reactivity as compared with those of healthy controls. We further demonstrate that IgG purified from eluates of RBCs of healthy donors induces agglutination of RBCs in an indirect Coombs assay to a similar extent as IgG purified from eluates of RBCs of patients with WAIHA. The capability of IgG to induce agglutination of RBCs is suppressed in unfractionated eluates of healthy donors' cells, whereas it is readily found in unfractionated eluates of patients' RBCs. IgM is an essential factor in controlling the ability of IgG in unfractionated RBC eluates to induce agglutination of RBCs. These observations indicate that anti-RBC IgG autoantibodies of patients with WAIHA share extensive similarity with natural antiRBC autoantibodies of healthy donors and suggest that defective control of IgG autoreactivity by autologous IgM is an underlying mechanism for autoimmune hemolysis in WAIHA. (Blood. 2000;95:328-335)
Surface expression of Rh-associated glycoprotein (RhAG) in nonerythroid COS-1 cells
Authors: Suyama Kimita , Li Hua , Zhu Alex
Published: October 2019

In the Rh blood system, RhAG (Rh-associated glycoprotein, or Rh50) is thought to be involved in Rh30 (D, CE) expression by forming a protein complex on the red cell surface. To obtain further insight into the Rh complex, we chose nonerythroid COS-1 cells instead of proerythroblast-like K562 cells, which produce endogenous Rh proteins as cell host, for the expression of both RhAG and RhD. The RhAG cDNA was subcloned into a retroviral vector, and a stable COS-1 cell line was then established via retroviral transduction. Surface expression of RhAG on the COS-1 cells was monitored by flow cytometry using mouse monoclonal anti-RhAG(2D10). Under these conditions, we detected significant expression of RhAG on the cell surface, compared to stable COS-1 cells transduced with the vector alone. To confirm the results, we isolated RhAG by immunoprecipitation from the lysate of the COS-1 cells, which were metabolically labeled with [35S]-methionine. A strong band of the 32 kd on SDS-PAGE was obtained, corresponding to the results obtained from other cultured cells (K562 cell and others), which always produce partially glycosylated RhAG with a molecular weight of 32 kd. Thus, RhAG was expressed without Rh30 and other Rh-related glycoproteins (LW, glycophorin B) in nonerythroid cells. Using the same strategy, however, we could not express RhD epitopes on COS-1 cells even in the presence of RhAG cDNA, suggesting that other factors might be required for the surface expression of RhD antigen. (Blood. 2000;95:336-341)
Single-tube multiplex-PCR screen for common deletional determinants of α-thalassemia
Authors: Chong Samuel S. , Boehm Corinne D. , Higgs Douglas R. , Cutting Garry R.
Published: October 2019

-Thalassemia is very common throughout all tropical and subtropical regions of the world. In Southeast Asia and the Mediterranean regions, compound heterozygotes and homozygotes may have anemia that is mild to severe (hemoglobin [Hb] H disease) or lethal (Hb Bart's hydrops fetalis). We have developed a reliable, single-tube multiplex–polymerase chain reaction (PCR) assay for the 6 most frequently observed determinants of -thalassemia. The assay allows simple, high throughput genetic screening for these common hematological disorders. (Blood. 2000;95:360-362)
Activation of C-C β-chemokines in human peripheral blood γδ T cells by isopentenyl pyrophosphate and regulation by cytokines
Authors: Cipriani Barbara , Borsellino Giovanna , Poccia Fabrizio , Placido Roberta , Tramonti Daniela , Bach Simona , Battistini Luca , Brosnan Celia F.
Published: October 2019

Human γδ T lymphocytes respond to viral, bacterial, protozoal, and tumoral antigens, but their precise function remains unknown. In adults the major circulating γδ T-cell subset expresses the Vγ9Vδ2 T-cell receptor and responds to protease-resistant phosphorylated derivatives found in many pathogens. In this study we show that activation of Vδ2+ cells with the nonpeptidic antigen isopentenyl pyrophosphate (IPP) rapidly induces (within 4-12 hours) the C-C chemokines MIP-1, MIP-1β, and lymphotactin but not MCP-1. The most robust response was obtained for MIP-1β. IPP induction of MIP-1 and MIP-1β was not affected by costimulation with interleukin-4 (IL-4), IL-10, TGF-β, or interferon-γ (INF-γ). However, IL-12 significantly enhanced IPP-induced expression and release of MIP-1 that was down-regulated by TGF-β whereas the induction of MIP-1β by IPP+IL-12 was refractory to cotreatment with TGFβ indicating that these chemokines are differentially regulated by these cytokines. Vδ2+ T cells also expressed a wide range of C-C chemokine receptors including CCR1, CCR5, and CCR8, all of which were down-regulated following activation. We conclude that Vδ2+ cells can be rapidly induced by components of bacterial cell walls to express high levels of proinflammatory chemokines, supporting an important role for these cells in the early stages of the inflammatory responses to many common pathogens. (Blood. 2000, 95:39-47)
Molecular heterogeneity in complete cytogenetic responders after interferon-α therapy for chronic myelogenous leukemia: low levels of minimal residual disease are associated with continuing remission
Authors: Hochhaus Andreas , Reiter Andreas , Reichert Susanne Saußele, Anja , Emig Michael , Kaeda Jaspal , Schultheis Beate , Berger Ute , Shepherd Patricia C. A. , Allan Norman C. , Hehlmann Rüdiger , Goldman John M. , Cross Nicholas C. P.
Published: October 2019

A substantial minority of patients with chronic myelogenous leukemia (CML) achieve a complete response (CR) to treatment with interferon- (IFN), defined as the disappearance of Philadelphia chromosome-positive metaphases. Currently it is unclear how long IFN treatment should be continued for such patients. We used a competitive reverse transcriptase-polymerase chain reaction (RT-PCR) to quantify levels of BCR-ABL transcripts in 297 peripheral blood specimens collected from 54 patients who had achieved CR with IFN. The median duration of observation was 1.9 years (range, 0.3-11.0 years). Total ABL transcripts were quantified as internal control and results were expressed as the ratio BCR-ABL/ABL. All 54 patients had molecular evidence of residual disease, although 3 patients were intermittently PCR negative. The median BCR-ABL/ABL ratio at the time of maximal response for each patient was 0.045% (range, 0%-3.6%). During the period of observation 14 patients relapsed, 11 cytogenetically to chronic phase disease and 3 directly to blastic phase. The median ratio of BCR-ABL/ABL at maximal response was significantly higher in patients who relapsed than in those who remained in CR (0.49% versus 0.021%,P < 0.0001). Our findings show that the level of residual disease falls with time in complete responders to IFN, but molecular evidence of disease is rarely if ever eliminated. The actual level of minimal residual disease correlates with the probability of relapse. We suggest that for patients who reach CR, IFN should be continued at least until relatively low levels of residual leukemia are achieved. (Blood. 2000;95:62-66)
Daclizumab, a humanized anti-interleukin-2 receptor alpha chain antibody, for treatment of acute graft-versus-host disease
Authors: Przepiorka D. , Kernan N. A. , Ippoliti C. , Papadopoulos E. B. , Giralt S. , Khouri I. , Lu J.-G. , Gajewski J. , Durett A. , Cleary K. , Champlin R. , Andersson B. S. , Light S.
Published: October 2019

Daclizumab, a humanized monoclonal IgG1 directed against the  chain of the interleukin-2 receptor (IL-2R), is a competitive inhibitor of IL-2 on activated lymphocytes. To test the hypothesis that specific inhibition of activated lymphocytes in patients with ongoing acute graft-versus-host disease (GVHD) might ameliorate the process, we treated 43 patients with advanced or steroid-refractory GVHD with daclizumab. The first cohort of 24 patients was treated with daclizumab 1 mg/kg on days 1, 8, 15, 22, and 29. On day 43, the complete response (CR) rate was 29% (95% confidence interval [CI], 13%-51%). Survival on day 120 was 29% (95% CI, 13%-51%). A second cohort of 19 patients was treated with daclizumab 1 mg/kg on days 1, 4, 8, 15, and 22. For these patients, the CR rate on day 43 was 47% (95% CI, 24%-71%), and survival on day 120 was 53% (95% CI, 29%-76%). There were no infusion-related reactions and no serious side effects related to daclizumab. Following treatment, there was a reduction in serum concentrations of soluble IL-2R and peripheral blood CD3 + 25+ lymphocytes, but these changes were not predictive of response. Daclizumab has substantial activity for the treatment of acute GVHD, and the second regimen evaluated is recommended for a controlled study. (Blood, 2000; 95:83-89)
Chemokine receptors and their role in inflammation and infectious diseases
Authors: Murdoch Craig , Finn Adam
Published: October 2019

Chemokines are small peptides that are potent activators and chemoattractants for leukocyte subpopulations and some nonhemopoietic cells. Their actions are mediated by a family of 7-transmembrane G-protein–coupled receptors, the size of which has grown considerably in recent years and now includes 18 members. Chemokine receptor expression on different cell types and their binding and response to specific chemokines are highly variable. Significant advances have been made in understanding the regulation of chemokine receptor expression and the intracellular signaling mechanisms used in bringing about cell activation. Chemokine receptors have also recently been implicated in several disease states including allergy, psoriasis, atherosclerosis, and malaria. However, most fascinating has been the observation that some of these receptors are used by human immunodeficiency virus type 1 in gaining entry into permissive cells. This review will discuss structural and functional aspects of chemokine receptor biology and will consider the roles these receptors play in inflammation and in infectious diseases.
Rituximab monoclonal antibody as initial systemic therapy for patients with low-grade non-Hodgkin lymphoma
Authors: Hainsworth John D. , Burris Howard A. , Morrissey Lisa H. , Litchy Sharlene , Scullin Daniel C. , Bearden James D. , Richards Paul , Greco F. Anthony
Published: October 2019

Rituximab, a chimeric antibody that targets CD20+ B cells, produces a 48% response rate in patients with refractory low-grade non-Hodgkin lymphoma. In this phase II trial, patients with low-grade non-Hodgkin lymphoma who had previously received no systemic therapy were treated with rituximab, 375 mg/m2, administered by IV infusion for 4 consecutive weeks. Patients with objective response or stable disease received repeat 4-week courses of rituximab at 6-month intervals. At the time of initial reevaluation at 6 weeks, 21 of 39 patients (54%) had objective response to treatment, and an additional 14 patients (36%) had stable disease or minor response. Response rates were similar in patients with follicular and small lymphocytic (CLL-type) lymphoma (52% versus 57%, respectively). At present, follow-up is short and only 13 patients have undergone a second course of rituximab treatment. However, 4 additional responses were documented either prior to the second course of rituximab (2 patients) or following the second course (2 patients) and 4 patients improved from partial to complete response. The current response rate is 64%, with 6 complete responses (15%). Treatment with rituximab was well tolerated, with only 1 patient experiencing grade 3/4 infusion-related toxicity. Rituximab is well tolerated and highly active in patients with low-grade non-Hodgkin lymphoma previously untreated with systemic therapy. Although further follow-up is required, the demonstration of minimal toxicity and considerable activity of this new biologic agent represents an important beginning of more specific, less toxic treatment for this important group of cancer patients.
Limiting numbers of G156A O6-methylguanine–DNA methyltransferase-transduced marrow progenitors repopulate nonmyeloablated mice after drug selection
Authors: Davis Brian M. , Koç Omer N. , Gerson Stanton L.
Published: October 2019

The limited efficacy of hematopoietic gene therapy can be improved by in vivo selection for transduced long-term repopulating cells (LTRC). We selected for G156A MGMT (▵MGMT) transduced LTRC present in 5 × 104 to 100 × 104 marrow cells infused into nonmyeloablated mice by the administration of O6-benzylguanine (BG) and BCNU every 3 to 4 weeks. To facilitate engraftment, mice were given a nonablative dose of BG and BCNU before infusion. Without selection, ▵MGMT was not detected in any hematopoietic colony-forming units (CFU) 24 to 30 weeks after infusion. After BG and BCNU, ▵MGMT+ CFU were frequently detected, and their proportions increased with each treatment cycle. After 2 to 3 cycles of BG and BCNU, many mice were stably reconstituted with 75% to 100% ▵MGMT+ CFU for at least 6 months, representing up to 940-fold enrichment. Thus, BG and BCNU stem cell toxicity allows ▵MGMT-transduced LTRC to repopulate the bone marrow. This degree of selection pressure in nonmyeloablated mice is far greater than that observed in previous drug-resistance gene transfer studies. These data support our approved clinical trial to select for drug-resistant, transduced hematopoietic cells, potentially decreasing cumulative drug-induced myelosuppression in patients with cancer. These data also suggest that ▵MGMT may be a potent, dominant, selectable marker for use in dual gene therapy.
Thrombocyte HLA molecules retain nonrenewable endogenous peptides of megakaryocyte lineage and do not stimulate direct allocytotoxicity in vitro
Authors: Gouttefangeas Cécile , Diehl Marianne , Keilholz Wieland , Hörnlein Rainer Frank , Stevanović Stefan , Rammensee Hans-Georg
Published: October 2019

The origin and the function of HLA class I molecules present on the surface of human platelets are still unclear. In particular, it is controversial which fraction of these class I molecules represents integral membrane components derived from the megakaryocyte-platelet lineage versus soluble plasma HLA molecules acquired by adsorption. Results of the present study show that HLA-A2 ligands isolated from platelets possess the same peptide motif as described for HLA-A2-associated peptides obtained from nucleated cells. Sequencing of these platelet-derived peptides reveals that they originate mainly from ubiquitously expressed proteins also present in the megakaryocyte-platelet lineage. Moreover, one of these peptides derives from the GPIX protein, which is specifically expressed by platelets and their precursors. Platelet HLA molecules are unstable in vitro at 37°C, but can be partially stabilized by addition of exogenous β2-microglobulin and HLA class I binding peptide, suggesting that platelets cannot load HLA molecules with endogenous peptides. In in vitro experiments platelets were used to stimulate peripheral blood mononuclear cells. No allospecific cytotoxicity was observed after primary stimulation, or secondary restimulation, with allogenic resting or activated platelets, even in the presence of additional third-party helper activity. These data indicate that HLA class I molecules from platelets cannot directly induce allogenic CD8+ cytotoxic T-cell response in vitro.
Deficiency of the Fas apoptosis pathway without Fas gene mutations is a familial trait predisposing to development of autoimmune diseases and cancer
Authors: Ramenghi Ugo , Bonissoni Sara , Migliaretti Giuseppe , DeFranco Simona , Bottarel Flavia , Gambaruto Caterina , DiFranco Daniela , Priori Roberta , Conti Fabrizio , Dianzani Irma , Valesini Guido , Merletti Franco , Dianzani Umberto
Published: October 2019

Fas/Apo-1 (CD95) triggers programmed cell death (PCD) and is involved in immune response control and cell-mediated cytotoxicity. In the autoimmune/lymphoproliferative syndrome (ALPS), inherited loss-of-function mutations of the Fas gene cause nonmalignant lymphoproliferation and autoimmunity. We have recently identified an ALPS-like clinical pattern (named autoimmune lymphoproliferative disease [ALD]) in patients with decreased Fas function, but noFas gene mutation. They also displayed decreased PCD response to ceramide, triggering a death pathway partially overlapping that used by Fas, which suggests that ALD is caused by downstream alterations of the Fas signaling pathway. Decreased Fas function is also involved in tumor development, because somatic mutations hitting the Fas system may protect neoplastic cells from immune surveillance. This work assessed the inherited component of the ALD defect by evaluating Fas- and ceramide-induced T-cell death in both parents and 4 close relatives of 10 unrelated patients with ALD. Most of them (22 of 24) displayed defective Fas- or ceramide-induced (or both) cell death. Moreover, analysis of the family histories showed that frequencies of autoimmunity and cancer were significantly increased in the paternal and maternal line, respectively. Defective Fas- or ceramide-induced T-cell death was also detected in 9 of 17 autoimmune patients from 7 families displaying more than a single case of autoimmunity within first- or second-degree relatives (multiple autoimmune syndrome [MAS] patients). Autoimmune diseases displayed by ALD and MAS families included several organ-specific and systemic forms. These data suggest that ALD is due to accumulation of several defects in the same subject and that these defects predispose to development of cancer or autoimmune diseases other than ALPS/ALD.
Chronic neutropenia mediated by Fas ligand
Authors: Liu Jin Hong , Wei Sheng , Lamy Thierry , Epling-Burnette P. K. , Starkebaum Gordon , Djeu Julie Y. , Loughran Thomas P.
Published: October 2019

Chronic neutropenia, often associated with rheumatoid arthritis, is a characteristic finding in large granular lymphocyte (LGL) leukemia. The mechanism of neutropenia is not known. Normal neutrophil survival is regulated by the Fas–Fas ligand apoptotic system. We hypothesized that neutropenia in LGL leukemia is mediated by dysregulated expression of Fas ligand. Levels of Fas ligand in serum samples from patients with LGL leukemia were measured with a Fas ligand enzyme-linked immunosorbent assay. The effects of serum from patients with LGL leukemia on apoptosis of normal neutrophils were determined by flow cytometry and morphologic assessment. High levels of circulating Fas ligand were detected in 39 of 44 serum samples from patients with LGL leukemia. In contrast, Fas ligand was undetectable in 10 samples from healthy donors. Serum from the patients triggered apoptosis of normal neutrophils that depended partly on the Fas pathway. Resolution of neutropenia was associated with disappearance or marked reduction in Fas ligand levels in 10 of 11 treated patients. These data suggest that high levels of Fas ligand are a pathogenetic mechanism in human disease.
The chemokine SDF-1 activates the integrins LFA-1, VLA-4, and VLA-5 on immature human CD34+ cells: role in transendothelial/stromal migration and engraftment of NOD/SCID mice
Authors: Peled Amnon , Kollet Orit , Ponomaryov Tanya , Petit Isabelle , Franitza Suzanna , Grabovsky Valentin , Slav Michal Magid , Nagler Arnon , Lider Ofer , Alon Ronen , Zipori Dov , Lapidot Tsvee
Published: October 2019

Hematopoietic stem cell homing and engraftment require several adhesion interactions, which are not fully understood. Engraftment of nonobese/severe combined immunodeficiency (NOD/SCID) mice by human stem cells is dependent on the major integrins very late activation antigen–4 (VLA-4); VLA-5; and to a lesser degree, lymphocyte function associated antigen–1 (LFA-1). Treatment of human CD34+cells with antibodies to either VLA-4 or VLA-5 prevented engraftment, and treatment with anti–LFA-1 antibodies significantly reduced the levels of engraftment. Activation of CD34+ cells, which bear the chemokine receptor CXCR4, with stromal derived factor 1 (SDF-1) led to firm adhesion and transendothelial migration, which was dependent on LFA-1/ICAM-1 (intracellular adhesion molecule–1) and VLA-4/VCAM-1 (vascular adhesion molecule–1). Furthermore, SDF-1–induced polarization and extravasation of CD34+/CXCR4+ cells through the extracellular matrix underlining the endothelium was dependent on both VLA-4 and VLA-5. Our results demonstrate that repopulating human stem cells functionally express LFA-1, VLA-4, and VLA-5. Furthermore, this study implies a novel approach to further advance clinical transplantation.
Vitamin A deficiency in mice causes a systemic expansion of myeloid cells
Authors: Kuwata Takeshi , Wang I-Ming , Tamura Tomohiko , Ponnamperuma Roshini M. , Levine Rachel , Holmes Kevin L. , Morse Herbert C. , De Luca Luigi M. , Ozato Keiko
Published: October 2019

To examine the role of retinoids in hematopoietic cell growth in vivo, we studied female SENCAR mice made vitamin A deficient by dietary restriction. Deficient mice exhibited a dramatic increase in myeloid cells in bone marrow, spleen, and peripheral blood. The abnormal expansion of myeloid cells was detected from an early stage of vitamin A deficiency and contrasted with essentially normal profiles of T and B lymphocytes. This abnormality was reversed on addition of retinoic acid to the vitamin A–deficient diet, indicating that the myeloid cell expansion is a direct result of retinoic acid deficiency. TUNEL analysis indicated that spontaneous apoptosis, a normal process in the life cycle of myeloid cells, was impaired in vitamin A–deficient mice, which may play a role in the increased myeloid cell population. Quantitative reverse transcriptase-polymerase chain reaction analysis of purified granulocytes showed that expression of not only RAR, but RXRs, 2 nuclear receptors that mediate biologic activities of retinoids, was significantly reduced in cells of deficient mice. This work shows that retinoids critically control the homeostasis of myeloid cell population in vivo and suggests that deficiency in this signaling pathway may contribute to various myeloproliferative disorders.
High-affinity binding to the GM-CSF receptor requires intact N-glycosylation sites in the extracellular domain of the β subunit
Authors: Niu Linghao , Heaney Mark L. , Vera Juan Carlos , Golde David W.
Published: October 2019

The human granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor consists of 2 glycoprotein subunits, GMR and GMRβ. GMR in isolation binds to GM-CSF with low affinity. GMRβ does not bind GM-CSF by itself, but forms a high-affinity receptor in association with GMR. Previously, it was found that N-glycosylation of GMR is essential for ligand binding. The present study investigated the role of N-glycosylation of the β subunit on GM-CSF receptor function. GMRβ has 3 potential N-glycosylation sites in the extracellular domain at Asn58, Asn191, and Asn346. Single mutants and triple mutants were constructed, converting asparagine in the target sites to aspartic acid or alanine. A single mutation at any of the 3 consensus N-glycosylation sites abolished high-affinity GM-CSF binding in transfected COS cells. Immunofluorescence and subcellular fractionation studies demonstrated that all of the GMRβ mutants were faithfully expressed on the cell surface. Reduction of apparent molecular weight of the triple mutant proteins was consistent with loss of N-glycosylation. Intact N-glycosylation sites of GMRβ in the extracellular domain are not required for cell surface targeting but are essential for high-affinity GM-CSF binding.