bluuebunny/crossref_metadata_2025 huggingface dataset:
Efficient retrovirus transduction of mouse pluripotent hematopoietic stem cells mobilized into the peripheral blood by treatment with granulocyte colony-stimulating factor and stem cell factor
Authors: Bodine DM , Seidel NE , Gale MS , Nienhuis AW , Orlic D
Published: October 2019

Cytokine-mobilized peripheral blood cells have been shown to participate in hematopoietic recovery after bone marrow (BM) transplantation, and are proposed to be useful targets for retrovirus- mediated gene transfer protocols. We treated mice with granulocyte colony-stimulating factor (G-CSF) and stem cell factor (SCF) to mobilize hematopoietic progenitor cells into the peripheral blood. These cells were analyzed for the number and frequency of pluripotent hematopoietic stem cells (PHSC). We found that splenectomized animals treated for 5 days with G-CSF and SCF showed a threefold increase in the absolute number of PHSC over normal mice. The number of peripheral- blood PHSC increased 250-fold from 29 per untreated mouse to 7,200 in peripheral-blood PHSC in splenectomized animals treated for 5 days with G-CSF and SCF. Peripheral blood PHSC mobilized by treatment with G-CSF and SCF were analyzed for their ability to be transduced by retroviral vectors. Peripheral-blood PHSC from splenectomized animals G-CSF and SCF were transduced with a recombinant retrovirus containing the human MDR-1 gene. The frequency of gene transfer into peripheral blood PHSC from animals treated for 5 and 7 days was two-fold and threefold higher than gene transfer into PHSC from the BM of 5-fluorouracil-treated mice (P < .01). We conclude that peripheral blood stem cells mobilized by treatment with G-CSF and SCF are excellent targets for retrovirus- mediated gene transfer.
The immunosuppressant rapamycin blocks in vitro responses to hematopoietic cytokines and inhibits recovering but not steady-state hematopoiesis in vivo
Authors: Quesniaux VF , Wehrli S , Steiner C , Joergensen J , Schuurman HJ , Herrman P , Schreier MH , Schuler W
Published: October 2019

The immunosuppressive drug rapamycin suppresses T-cell activation by impairing the T-cell response to lymphokines such as interleukin-2 (IL- 2) and interleukin-4 (IL-4). In addition, rapamycin blocks the proliferative response of cell lines to a variety of hematopoietic growth factors, including interleukin-3 (IL-3), interleukin-6 (IL-6), granulocyte-colony stimulating factor (G-CSF), granulocyte macrophage- colony stimulating factor (GM-CSF), and kit ligand (KL), suggesting that it should be a strong inhibitor of hematopoiesis. In this report, we studied the effects of rapamycin on different hematopoietic cell populations in vitro and in vivo. In vitro, rapamycin inhibited the proliferation of primary bone marrow cells induced by IL-3, GM-CSF, KL, or a complex mixture of factors present in cell-conditioned media. Rapamycin also inhibited the multiplication of colony-forming cells in suspension cultures containing IL-3 plus interleukin-1 (IL-1) or interleukin-11 (IL-11) plus KL. In vivo, treatment for 10 to 28 days with high doses of rapamycin (50 mg/kg/d, orally) had no effect on myelopoiesis in normal mice, as measured by bone marrow cellularity, proliferative capacity, and number of colony-forming progenitors. In contrast, the same treatment strongly suppressed the hematopoietic recovery normally seen 10 days after an injection of 5-fluorouracil (5- FU; 150 mg/kg, intravenously [i.v.]). Thus, rapamycin may be detrimental in myelocompromised individuals. In addition, the results suggest that the rapamycin-sensitive cytokine-driven pathways are essential for hematopoietic recovery after myelodepression, but not for steady-state hematopoiesis.
Human CD34+ fetal liver stem cells differentiate to T cells in a mouse thymic microenvironment
Authors: Plum J , De Smedt M , Defresne MP , Leclercq G , Vandekerckhove B
Published: October 2019

Hematopoietic stem cells differentiate in the thymus to T cells along precisely defined intermediates. This process is thymic epithelium dependent and involves cytokines and cell-cell interactions between thymic stroma and T-cell precursors. Here we report that highly purified human CD34++ fetal liver stem cells differentiate to mature T cells, when seeded into isolated fetal thymic lobes of severe combined immunodeficient mice, and subsequently cultured in vitro. The human stem cells differentiate sequentially into CD4+CD8-CD3-, CD4+CD8+CD3-, CD4+CD8+CD3+, and finally, CD4+CD8-CD3+4 and CD4-CD8+CD3++ cells. Phenotypic analysis for additional maturation markers showed that these CD4 and CD8 single-positive thymocytes are fully maturate cells. By immunochemistry, human HLA-DR+ cells with a dendritic morphology could be detected. This novel chimeric human-mouse fetal thymus organ culture offers a tool to study human T-cell ontogeny in vitro and is a rapid and reliable test method for T-cell precursor activity of cultured or transfected human stem cells.
Merozoite surface protein-3: a malaria protein inducing antibodies that promote Plasmodium falciparum killing by cooperation with blood monocytes
Authors: Oeuvray C , Bouharoun-Tayoun H , Gras-Masse H , Bottius E , Kaidoh T , Aikawa M , Filgueira MC , Tartar A , Druilhe P
Published: October 2019

We have previously found that the acquired protection against malaria implicates a mechanism of defense that relies on the cooperation between cytophilic antibodies and monocytes. Accordingly, an assay of antibody-dependent cellular inhibition (ADCI) of parasite growth was used as a means of selecting for molecules capable of inducing protective immunity to malaria. This allowed us to identify in the sera of clinically protected subjects an antibody specificity that promotes parasite killing mediated by monocytes. This antibody is directed to a novel merozoite surface protein (MSP-3) of a molecular mass of 48 kD. Purified IgG from protected subjects are effective in ADCI and those directed against MSP-3 are predominantly cytophilic. In contrast, in nonprotected individuals, whose antibodies are not effective in ADCI, anti-MSP-3 antibodies are mostly noncytophilic. A region in MSP-3 targetted by antibodies effective in the ADCI assay was identified and its sequence was determined; it contains an epitope not defined by a repetitive structure and does not appear to be polymorphic. Antibodies raised in mice against a peptide containing this epitope, as well as human antibodies immunopurified on this peptide, elicit a strong inhibition of Plasmodium falciparum growth in ADCI assay, whereas control antibodies, directed to peptides from other molecules, do not. The correlation between isotypes of antibodies produced against the 48- kD epitopes, clinical protection, and the ability of specific anti-MSP- 3 antibodies to block the parasite schizogony in the ADCI assay suggests that this molecule is involved in eliciting protective mechanisms.
Human HL-60 myeloid leukemia cells transport dehydroascorbic acid via the glucose transporters and accumulate reduced ascorbic acid
Authors: Vera JC , Rivas CI , Zhang RH , Farber CM , Golde DW
Published: October 2019

The cellular accumulation of vitamin C, a substance critical to human physiology, is mediated by transporters located at the cell membrane, and is regulated in a cell-specific manner. Neoplastic cells may have special needs for vitamin C. Therefore, we investigated the transport of vitamin C in a human myeloid leukemia cell line (HL-60). The HL-60 cells lacked the capacity to transport the reduced form of vitamin C, ascorbic acid, but they showed a remarkable ability to transport the oxidized form of vitamin C, dehydroascorbic acid (DHA). Uptake- accumulation studies indicated that the HL-60 cells accumulated ascorbic acid when provided with DHA. Kinetic analysis showed the presence of two functional activities involved in the uptake of DHA, one with low affinity and one with high affinity. Cytochalasin B and phloretin, which inhibit the passage of glucose through the facilitative glucose transporters, also inhibited the transport of DHA by HL-60 cells. Transport of DHA was completed by D- but not L-hexoses, and was sensitive to D-hexose-dependent counter transport acceleration. These data support the concept that HL-60 myeloid leukemic cells transport DHA through the facilitative hexose transporters (glucose transporters) and accumulate the reduced form of ascorbic acid.
Characterization of the human neutrophil C1q receptor and functional effects of free ligand on activated neutrophils
Authors: Eggleton P , Ghebrehiwet B , Coburn JP , Sastry KN , Zaner KS , Tauber AI
Published: October 2019

The partial characterization and expression of the C1q receptor (C1q-R) in relation to other complement receptors present on the surface of neutrophils has been examined, as well as the effects of free C1q on cell function. A polyclonal anti-C1q-R antibody recognizes a 68-kD neutrophil surface protein. C1q-R expression was not upregulated upon warming, priming, or exposure to FMLP, but decreased after exposure to phorbol myristate acetate (PMA), because of shedding of the receptor into the extracellular medium, as detected by enzyme-linked immunosorbent assay. CR3 and CR1 expression was upregulated from intracellular pools after cell stimulation by PMA. No evidence of intracellular pools of C1q-R was found, as assessed by immunoblotting of subcellular fractions. But C1q-R appeared to be expressed early in cell differentiation, was detected on undifferentiated HL-60 cells, and like CR3 expression, increased upon 5 days differentiation towards a neutrophil lineage. However, C1q-R expression decreased upon additional culture, whereas CR3 expression continued to increase. A large variation in the percentage of peripheral cells expressing C1q receptors in donors was observed, ranging from 13% to 100%, contrasting with CR3 receptors that exhibited less variability. Interactions between free monomeric C1q and neutrophils were also studied. Incubation of stimulated neutrophils with 10 to 100 micrograms/mL C1q resulted in a further increase in CR3 expression and adherence to albumin-coated surfaces. Staphylococci opsonized with low quantities of C1q (0.1 to 1 microgram/mL) mediated a moderate and sustained respiratory burst in neutrophils, whereas a burst of similar magnitude was generated only with free C1q at concentrations 10- to 100-fold higher. Stimulation was only partially inhibited if cells were first treated with anti-C1q-R antibody, suggesting other C1q binding proteins may be present on the cell surface. In summary, neutrophil C1q receptor is approximately 68-kD, exhibits varying expression on different subjects, and is not upregulated from intracellular stores on exposure to soluble stimuli. Stimulated, but not resting, neutrophils selectively respond to raised levels of free C1q, resulting in altered cell function and enhanced CR3 receptor expression. These studies thus suggest complex roles for C1q in neutrophil function.
Platelet alpha-granule and plasma membrane share two new components: CD9 and PECAM-1
Authors: Cramer EM , Berger G , Berndt MC
Published: October 2019

CD9 (p24) and PECAM1 (CD31) antigens are well-defined components of the platelet plasma membrane. Both are integral glycoproteins (GPs) implicated in the adhesive and aggregative properties of human platelets. In the present report, we have investigated their subcellular localization using immunoelectron microscopy. The monospecificity of the two polyclonal antibodies used was confirmed by immunoblotting. On normal resting platelets, immunolabeling for CD9 and PECAM1 was found lining the plasma membrane and the luminal face of the open canalicular system. Some labeling was also consistently found on the alpha-granule limiting membrane. This was confirmed by double labeling experiments in which fibrinogen and von Willebrand factor (vWF) were used as alpha-granule markers. CD9 and PECAM-1 were found lining the membrane of the same granules that contained fibrinogen and vWF in their matrix. CD9 and PECAM-1 thus appear to have an intracellular distribution identical to GPIIb-IIIa, a major aggregation platelet receptor. To rule out a cross-reactivity of the two polyclonal antibodies with GPIIb/IIIa, we studied PECAM1 and CD9 expression on the platelets from a patient with type I Glanzmann's thrombasthenia whose platelets are devoid of GPIIb/IIIa. The same pattern of labeling was observed for both antigens as for normal platelets. Normal platelets were further observed after stimulation by agonists that either fail to induce (ADP) or induce granule secretion (thrombin). After treatment with ADP, platelets changed shape and centralized their granules; the plasma membrane immunolabeling remained unchanged; and gold particles were still found decorating the periphery of the centralized alpha- granules. After thrombin treatment, alpha-granules fused with the platelet membrane and secretion occurred. A significant increase of labeling was then observed on the platelet surface. From these results we conclude that the alpha-granule membrane contains two additional receptors in common with the plasma membrane. This suggests that alpha- granule membrane receptors may originate from a dual mechanism: direct targeting from the Golgi complex in megakaryocytes (for alpha-granule- specific receptors such as P-selectin) or by endocytosis from the plasma membrane (for proteins distributed in the two compartments).
ENL, the gene fused with HRX in t(11;19) leukemias, encodes a nuclear protein with transcriptional activation potential in lymphoid and myeloid cells
Authors: Rubnitz JE , Morrissey J , Savage PA , Cleary ML
Published: October 2019

Chromosome band 11q23 is the site of recurring translocations with a variety of partner chromosomes in myeloid and lymphoid acute leukemias, infant leukemias, and treatment-induced secondary acute myelogenous leukemia. The translocation breakpoints cluster in a restricted region of the HRX gene resulting in fusion genes that encode an N-terminal portion of Hrx fused to various partner proteins. We have characterized the transcriptional transactivation properties of Enl, a protein that is fused to Hrx in t(11;19) leukemias. Enl is a nuclear protein that is capable of activating transcription from synthetic reporter genes in both lymphoid and myeloid cells, as well as in yeast. Deletion mutagenesis demonstrated that the minimal portion of Enl required for activation of transcription was localized to its C-terminal 90 amino acids. This region is highly conserved between Enl and the t(9;11) fusion partner Af-9 and is retained in all Hrx-Enl and Hrx-Af9 fusion proteins. Thus, the leukemogenic contribution and transcriptional activation potential of Enl colocalize to its highly conserved carboxy terminus, suggesting that Hrx-Enl chimeric proteins mediate alterations in the transcription program of t(11;19)-bearing cells.
Role for low-affinity receptor for IgE (CD23) in normal and leukemic B- cell proliferation
Authors: Fournier S , Rubio M , Delespesse G , Sarfati M
Published: October 2019

CD23 gene is overexpressed and abnormally regulated in the most frequent adult leukemic disorder, B chronic lymphocytic leukemia (B- CLL). Switch on and off in the upregulation of surface CD23 expression consistently occurs in the early stage of normal B-cell activation, suggesting a key role for CD23 in this process. We show here that, after ligation of mlg in the presence of interleukin-4, the increase of CD23 protein precedes B-cell DNA synthesis and mainly results from the strong induction of CD23 type-B isoform. Exposure of normal B cells to conventional or phosphorothioate-derivatized CD23 antisense oligonucleotides (predominantly type B) significantly augments B-cell proliferation induced by antigen receptor stimulation or direct contact with activated T cells. Unexpectedly, CD23 antisense, but not sense, oligonucleotides specifically enhance rather than suppress CD23 expression on B cells. Finally, a selective increase in CD23 type-B expression provokes the entry of resting (Go) CLL B cells into G1 and S phase of the cell cycle in the absence of any other stimulus, whereas it synergizes with tumor necrosis factor-alpha to increase the number of activated B cells. These results provide compelling evidence that CD23 represents an important molecule directly involved in the process of normal or leukemic B-cell activation and growth.
Erythroid-specific processing of human beta spectrin I pre-mRNA
Authors: Chu ZL , Wickrema A , Krantz SB , Winkelmann JC
Published: October 2019

Erythroid cells express a unique form of beta spectrin I as a result of tissue-specific alternative pre-mRNA processing. Nonerythroid cells that express the beta spectrin I gene include four additional exons at the 3′ end of the mature transcript, leading to elongation of the carboxyl terminus of the protein. The nonerythroid beta spectrin I isoform is not present in the red blood cell membrane skeleton; the erythroid isoform is not detected in other cell types. Therefore, developing erythroid cells acquire this tissue-specific pre-mRNA processing activity during differentiation. In the present study, we investigated the developmental timing of erythroid-specific pre-mRNA processing in human erythroid precursors. Partially purified human peripheral blood burst forming uniterythroid (BFU-E) cells were grown in culture for 5 to 12 days. beta Spectrin I mRNA transcripts were analyzed at different time points by S1 nuclease mapping. The processing of beta spectrin I transcripts was found to be exclusively erythroid from day 5 onward, indicating that erythroid-specific processing is not linked temporally to assembly of the mature erythroid membrane skeleton. Human erythroleukemia (HEL) cells had both erythroid and nonerythroid transcripts, indicating that both processing patterns can coexist. Induction of erythroid differentiation in HEL cells using hemin resulted in a partial switch toward the erythroid processing pattern of beta spectrin I transcripts. Using a genomic S1 probe that spans the erythroid polyadenylation signal, we found that a substantial portion of the transcripts detected by the erythroid cDNA S1 probe (in both cultured BFU-E and HEL cells) is incompletely processed pre-mRNA precursors. Poly(A) RNA selection before S1 analysis showed that the unprocessed transcripts are not polyadenylated. We conclude that (1) erythroid-specific pre-mRNA processing activity is present early in erythroid differentiation; (2) beta spectrin I transcripts that are unprocessed at the 3′ end accumulate, awaiting either erythroid or nonerythroid processing pathways, from which observation we infer that the regulated alternative pathways are both inefficient; and (3) HEL cells offer a human cell culture model in which to study the balance between the two pre-mRNA processing pathways. We speculate that erythroid cells evolved this tissue-specific pre-mRNA processing machinery for other erythroid genes in addition to beta spectrin I.
Fetal hemoglobin and potassium in isolated transferrin receptor- positive dense sickle reticulocytes
Authors: Franco RS , Barker-Gear R , Miller MA , Williams SM , Joiner CH , Rucknagel DL
Published: October 2019

A subset of sickle cells have an increased density at the reticulocyte stage of development, indicating that they are either abnormally dense upon release from the bone marrow or become dense quickly in the circulation. These cells are of interest because they most likely have severely disrupted cation regulation and a short lifespan. Based on the distribution of fetal hemoglobin (HbF) in the density fractions of sickle red blood cells (RBCs) and in vitro studies of cellular K+ loss, it seems likely that HbF content is an important in vivo determinant of dense cell formation. In this study, we tested the hypothesis that young, dense cells have low HbF content. Sickle RBCs were first separated into light and dense fractions. Reticulocytes were isolated from unfractionated cells and from each density fraction with an immunomagnetic technique directed against transferrin receptors (TfR) and assayed for the percentage of HbF and K+/Hb ratio. TfR+ reticulocytes isolated from unfractionated cells had a much lower HbF content when compared with all the unfractionated RBCs. This is most likely caused by enrichment of F cells because of a longer circulation life span. Heavy TfR+ reticulocytes had a K+/Hb ratio similar to that measured in the entire dense population and contained very low levels of HbF, averaging 2.5% of the level in all RBCs, 11.7% of the level in all TfR+ reticulocytes, and 4.0% of the level in all dense RBCs. These findings suggest that TfR+ dense cells derive predominantly from non-F cells. Furthermore, the amount of HbF in the circulating dense cells suggests that many of these cells do not derive from the TfR+ dense cells.
Hemagglutination assays for the diagnosis and prevention of IgA anaphylactic transfusion reactions
Authors: Sandler SG , Eckrich R , Malamut D , Mallory D
Published: October 2019

Passive hemagglutination assays (PHA) may be used to detect IgA antibodies to confirm clinical diagnoses of suspected IgA anaphylactic transfusion reactions. Passive hemagglutination inhibition assays (PHIA) may be used to identify IgA-deficient blood donors whose plasma- containing components are transfused to prevent anaphylactic transfusion reactions in prospective recipients at risk because of the presence of IgA antibodies. Using a standard PHA, we detected class- specific anti-IgA in 76.3% of 80 IgA-deficient patients with a history of an anaphylactic transfusion reaction, and in 21.7% of 97 asymptomatic IgA-deficient blood donors or their IgA-deficient family members. Using PHIA, we confirmed IgA deficiency (< 0.05 mg/dL) for the donors of 525 plasma-containing blood components that were transfused without acute clinical reactions to 48 IgA-deficient recipients with anti-IgA and/or a history of an anaphylactic transfusion reaction. The frequency of IgA-deficiency with class-specific anti-IgA among 32,376 random blood donors was 0.08% (1/1,200). The combined use of PHA for detecting anti-IgA and PHIA for measuring IgA concentration provides an effective and safe strategy for the diagnosis and prevention of IgA anaphylactic transfusion reactions. However, PHA for anti-IgA lacks specificity for identifying persons who are truly at risk for significant anaphylactic transfusion reactions. The consequence is an overdiagnosis of IgA anaphylactic transfusion reactions and an overestimation of the number of persons at risk for IgA anaphylactic transfusion reactions because of the detection of an IgA antibody in their serum.
Alternative, out-of-frame runt/MTG8 transcripts are encoded by the derivative (8) chromosome in the t(8;21) of acute myeloid leukemia M2
Authors: Tighe JE , Calabi F
Published: October 2019

In the t(8;21) of acute myeloid leukemia (AML) M2, breakpoints are clustered on both chromosomes. The chromosome 21 breakpoint cluster region (bcr) falls within the runt locus, in the intron immediately downstream of the exons encoding an evolutionary conserved domain (the runt box). Transcripts in which the runt box is fused in frame to a novel sequence derived from chromosome 8 (MTG8) have been previously identified and have been assumed to constitute a critical leukemogenic event. Here we show physical linkage of the chromosome 8 bcr to the MTG8 locus. Unexpectedly, not only does the bcr map upstream of the most 5′ MTG8 exon found in runt/MTG8 fusion transcripts, but it also maps upstream of a further 5′ exon. In addition, we demonstrate the presence of alternative transcripts, originating from the der(8) chromosome, in which runt is out of frame with MTG8. Thus, runt truncation per se, rather than its fusion to MTG8, may be the crucial leukemogenic event.
Sequential administration of recombinant human interleukin-3 and granulocyte-macrophage colony-stimulating factor after autologous bone marrow transplantation for malignant lymphoma: a phase I/II multicenter study
Authors: Fay JW , Lazarus H , Herzig R , Saez R , Stevens DA , Collins RH Jr , Pineiro LA , Cooper BW , DiCesare J , Campion M
Published: October 2019

Preclinical studies of recombinant human interleukin-3 (rhIL-3) and granulocyte-macrophage colony-stimulating factor (rhGM-CSF) have shown enhancement of multilineage hematopoiesis when administered sequentially. This study was designed to evaluate the safety, tolerability, and biologic effects of sequential administration of rhIL- 3 and rhGM-CSF after marrow ablative cytotoxic therapy and autologous bone marrow transplantation (ABMT) for patients with malignant lymphoma. Thirty-seven patients (20 patients with non-Hodgkin's lymphoma and 17 patients with Hodgkin's disease) received one of four different treatment regimens before ABMT. Patients were entered in one of four study groups to receive rhIL-3 (2.5 or 5.0 micrograms/kg/day) administered by subcutaneous injection for either 5 or 10 days starting 4 hours after the marrow infusion. Twenty-four hours after the last dose of rhIL-3, rhGM-CSF (250 micrograms/m2/d as a 2-hour intravenous infusion) administration was initiated. rhGM-CSF was administered daily until the absolute neutrophil count (ANC) was > or = 1,500/microL for 3 consecutive days or until day 27 posttransplant. The most frequent adverse events in the trial included nausea, fever, diarrhea, mucositis, vomiting, rash, edema, chills, abdominal pain, and tachycardia. Three patients were removed from the study because of chest, skeletal, and abdominal pain felt to be probably related to study drug. Four patients died during the study period because of complications unrelated to either rhIL-3 or rhGM-CSF. The median time to recovery of neutrophils (ANC > or = 500/microL) and platelets (platelet count > or = 20,000/microL) was 14 and 15 days, respectively. There were fewer days of platelet transfusions than seen in historical control groups using rhGM-CSF, rhG-CSF, or rhIL-3 alone. In addition, there were fewer days of red blood cell transfusions compared with historical controls using no cytokines or rhGM-CSF. These data indicate that the sequential administration of rhIL-3 and rhGM-CSF after ABMT is safe and generally well-tolerated and results in rapid recovery of multilineage hematopoiesis.
Treatment of large granular lymphocyte leukemia with oral low-dose methotrexate
Authors: Loughran TP Jr , Kidd PG , Starkebaum G
Published: October 2019

Morbidity and mortality in patients with T large granular lymphocyte (T- LGL) leukemia result from infections acquired during severe neutropenia. Optimum treatment for severe neutropenia remains undefined. We conducted an uncontrolled but prospective study of low- dose oral methotrexate, up to 10 mg/m2 weekly, in 10 patients with this disease. Therapeutic response was assessed by serial clinical evaluations and laboratory determinations including complete blood counts, lymphocyte phenotyping, and T-cell receptor gene rearrangement studies. A partial response was defined as a sustained increase in neutrophil count greater than 500/microL. A complete clinical remission was defined as achievement of a normal complete blood count and CD3+ LGL count. Previous prednisone treatment in eight of these patients had produced one clinical remission and four partial responses; tapering of prednisone in each of these patients resulted in recurrence of severe neutropenia. Five patients in this study received both methotrexate and tapering doses of prednisone. Complete clinical remissions on methotrexate were observed in five patients; an additional patient had a partial response. Molecular analyses of T-cell receptor gene rearrangement could not detect the abnormal clone in three of five patients achieving a complete clinical remission. Two weeks to 4 months of therapy were needed before attaining a neutrophil count greater than 500/microL. Complete and partial responses have been maintained on therapy, with a follow-up period ranging from 1.3 to 9.6 years. Low- dose oral methotrexate therapy is an effective treatment for some patients with LGL leukemia.
Distinct and overlapping direct effects of macrophage inflammatory protein-1 alpha and transforming growth factor beta on hematopoietic progenitor/stem cell growth
Authors: Keller JR , Bartelmez SH , Sitnicka E , Ruscetti FW , Ortiz M , Gooya JM , Jacobsen SE
Published: October 2019

Both transforming growth factor beta (TGF beta) and macrophage inflammatory protein 1 alpha (MIP-1 alpha) have been shown to be multifunctional regulators of hematopoiesis that can either inhibit or enhance the growth of hematopoietic progenitor cells (HPC). We report here the spectrum of activities of these two cytokines on different hematopoietic progenitor and stem cell populations, and whether these effects are direct or indirect. MIP-1 alpha enhances interleukin-3 (IL- 3)/and granulocyte-macrophage colony-stimulating factor (GM- CSF)/induced colony formation of normal bone marrow progenitor cells (BMC) and lineage-negative (Lin-) progenitors, but has no effect on G- CSF or CSF-1/induced colony formation. Similarly, TGF beta enhances GM- CSF/induced colony formation of normal BMC and Lin- progenitors. In contrast, TGF beta inhibits IL-3/ and CSF-1/induced colony formation of Lin- progenitors. The effects of MIP-1 alpha and TGF beta on the growth of Lin- progenitors were direct and correlate with colony formation in soft agar. Separation of the Lin- cells into Thy-1 and Thy-1lo subsets showed that the growth of Thy-1lo Lin- cells is directly inhibited by MIP-1 alpha and TGF beta regardless of the cytokine used to stimulate growth (IL-3), GM-CSF, or CSF-1). In contrast, two other stem cell populations (0% to 15% Hoechst 33342/Rhodamine 123 [Ho/Rh123] and Lin- Sca-1+ cells) were markedly inhibited by TGF beta and unaffected by MIP- 1 alpha. Furthermore, MIP-1 alpha has no effect on high proliferative potential colony-forming cells 1 or 2 (HPP-CFC/1 or /2) colony formation in vitro, whereas TGF beta inhibits both HPP-CFC/1 and HPP- CFC/2. Thus, MIP-1 alpha and TGF beta are direct bidirectional regulators of HPC growth, whose effects are dependent on other growth factors present as well as the maturational state of the HPC assayed. The spectrum of their inhibitory and enhancing activities shows overlapping yet distinct effects.
Molecular analysis of Polish patients with factor VII deficiency
Authors: Arbini AA , Bodkin D , Lopaciuk S , Bauer KA
Published: October 2019

We analyzed the mutations in patients from 10 Polish kindreds with a bleeding diathesis due to factor VII deficiency. Patients from eight families had plasma levels of factor VII coagulant activity (VII:C) and factor VII antigen (VII:Ag) that were less than 4% of normal. The coding sequence of the factor VII gene was amplified from genomic DNA by polymerase chain reaction (PCR). Sequencing demonstrated a C to T transition at position 10798 resulting in Ala294Val, a G to A transition at 10976 resulting in Arg353Gln, and a single bp deletion at 11125 to 11128 causing a frameshift mutation in the triplet encoding amino acid 404. Homozygosity for the three sequence alterations was confirmed with the restriction enzymes AvaII and MspI and allele specific PCR, respectively. A homozygous patient from a ninth family with levels of VII:C and VII:Ag of 4% and 17%, respectively, had Ala294Val and the frameshift mutation, but not Arg353Gln. Investigation of a homozygous patient from a tenth kindred with VII:C and VII:Ag of 11% and 47%, respectively, demonstrated Ala294Val and Arg353Gln, but not the frameshift mutation. Based on the above data, we conclude that the frameshift mutation in the codon for amino acid 404 is associated with marked reductions in VII:C, Arg353Gln can decrease plasma levels of factor VII in the presence of other mutations in the factor VII gene, and Ala294Val results in a dysfunctional factor VII molecule.
Lymphocyte depletion during treatment with intensive chemotherapy for cancer
Authors: Mackall CL , Fleisher TA , Brown MR , Magrath IT , Shad AT , Horowitz ME , Wexler LH , Adde MA , McClure LL , Gress RE
Published: October 2019

Recently we have observed an increased incidence of opportunistic infections in patients treated with intensive chemotherapy for cancer. Because T-cell depletion is associated with similar clinical events in human immunodeficiency virus infection and after bone marrow transplantation, we have analyzed peripheral blood lymphocyte populations in a series of patients during treatment with intensive chemotherapy for cancer. Although neutrophil, monocyte, and platelet numbers consistently recovered to greater than 50% of pretreatment values after each sequential cycle of therapy, lymphocyte numbers did not recover within the same time period. B cells decreased rapidly from a mean value of 149 +/- 46/mm3 before chemotherapy to 4 +/- 1/mm3 during chemotherapy (P = .01). CD4+ T cells decreased from a mean of 588 +/- 76/mm3 before chemotherapy to 105 +/- 28/mm3 during chemotherapy (P = .0002) and CD8+ T cells decreased from a mean of 382 +/- 41/mm3 before chemotherapy to 150 +/- 46/mm3 during chemotherapy (P = .0009). Natural killer cell numbers did not show significant declines (171 +/- 30/mm3 before, 114 +/- 24/mm3 during, P = .19). Based on the history of opportunistic complications in patients with other disorders who display similar degrees of CD4+ T-cell lymphopenia and preliminary observations in this population, immune incompetence could surface as a dose-limiting toxicity for highly dose-intensive chemotherapy regimens.
Histamine induces interleukin-8 secretion by endothelial cells
Authors: Jeannin P , Delneste Y , Gosset P , Molet S , Lassalle P , Hamid Q , Tsicopoulos A , Tonnel AB
Published: October 2019

It has been shown that histamine induces early changes on endothelial cells (EC), such as a transient expression of P-selectin and secretion and/or surface expression of early mediators (eg, prostacyclin [PG1(2)], platelet-activating factor [PAF], and leukotriene B4 [LTB4]). However, delayed effects of histamine on EC and particularly on cytokine production are undefined. In this study, the effect of histamine on interleukin (IL)-8 production by EC was evaluated using an enzyme-linked immunosorbent assay (ELISA) method and mRNA expression. The results showed that histamine increased the secretion and the mRNA expression of IL-8 by EC. Histamine-induced IL-8 production was (1) dose-dependent (at a dose > or = 10(-6) mol/L), (2) potentialized by costimulation with tumor necrosis factor (TNF)-alpha, (3) inhibited by H1 or H2 histamine receptor antagonists, and (4) significantly increased 4 hours after the initial stimulation. These data suggest that histamine may be involved in the control of the late inflammatory reaction associated to allergic disorders through IL-8 secretion by EC.
Identification of upstream signals regulating interleukin-6 gene expression during in vitro treatment of human B cells with pokeweed mitogen
Authors: Chauhan D , Kharbanda S , Uchiyama H , Urashima M , Fragoso R , Sen J , Kufe DW , Anderson KC
Published: October 2019

The present studies have characterized the regulation of interleukin-6 (IL-6) gene expression during pokeweed mitogen (PWM)-driven human B- cell differentiation. PWM induced an early and transient increase in the expression of immediate-early response genes of the jun/fos leucine zipper family (c-jun, jun B, c-fos, and fos-B). The induction of c-jun mRNA by PWM was concentration dependent. Nuclear run-on assays showed that PWM treatment is associated with an increased rate of c-jun gene transcription. The induction of c-jun mRNA precedes the induction of IL- 6 gene expression and IL-6 secretion by the B cells. c-Jun antisense, but not sense, oligodeoxynucleotide (ODN) significantly decreases PWM- related B-cell (1) proliferation; (2) IL-6 mRNA induction; (3) IL-6 secretion; and (4) nuclear extract binding to AP-1 in electrophoretic mobility shift assay. In contrast, c-Fos anti-sense ODN did not effect either IL-6 mRNA induction or IL-6 secretion triggered in B cells by PWM. The results further show activation of c-Raf-1 kinase in PWM- treated B cells. Raf-1 acts upstream to mitogen-activated protein (MAP) kinase; therefore, studies were performed to assay for MAP kinase activation in these cells. The results show an increase in phosphorylation of myelin basic protein (MBP) and c-Jun “Y” peptide in PWM-treated B cells. Taken together, these findings suggest that PWM is able to initiate an intracytoplasmic signaling cascade in normal human splenic B cells, which, at least in part, involves serine/threonine protein kinases. These results show transient induction of immediate- early response genes in B cells and support a potential role for the c- jun gene product in regulation of IL-6 transcription and secretion.
Interleukin-12 is chemotactic for natural killer cells and stimulates their interaction with vascular endothelium
Authors: Allavena P , Paganin C , Zhou D , Bianchi G , Sozzani S , Mantovani A
Published: October 2019

We investigated the chemotactic activity of interleukin (IL)-12 on human natural killer (NK) cells and other leukocyte subsets. It was found that IL-12 induced directional migration of highly enriched preparations of NK cells (> 80% CD16+ and CD56+) and CD3-activated T cells (both of CD4 and CD8 subset), but not resting T cells and monocytes. On the contrary, purified polymorphonuclear cells (PMN) showed significant and reproducible chemotactic response to IL-12. The effects of IL-12 on leukocyte migration were observed in a narrow concentration range with a peak at approximately 7.5 ng/mL, and were abrogated by monoclonal antibody (MoAb) anti-IL-12 or after cytokine boiling. We also investigated the interaction of NK cells with vascular endothelium in vitro. Overnight treatment of NK cells with IL-12 augmented their binding to cultured endothelial cells (EC) obtained from umbilical veins. IL-12-increased binding was better observed when resting rather than IL-1-activated EC were used as substratum of adhesion. IL-12-augmented binding of NK cells to resting or IL-1- activated EC involved the LFA-1/ICAM-1 and VLA-4/VCAM-1 pathways. Thus, by inducing migration and interaction with EC, IL-12 regulates crucial determinants of NK-cell recruitment in tissues.
Primary tumor cells of myeloma patients induce interleukin-6 secretion in long-term bone marrow cultures
Authors: Lokhorst HM , Lamme T , de Smet M , Klein S , de Weger RA , van Oers R , Bloem AC
Published: October 2019

Long-term bone marrow cultures (LTBMC) from patients with multiple myeloma (MM) and normal donors were analyzed for immunophenotype and cytokine production. Both LTBMC adherent cells from myeloma and normal donor origin expressed CD10, CD13, the adhesion molecules CD44, CD54, vascular cell adhesion molecule 1, very late antigen 2 (VLA-2), and VLA- 5, and were positive for extracellular matrix components fibronectin, laminin, and collagen types 3 and 4. LTBMC from myeloma patients and normal donors spontaneously secreted interleukin-6 (IL-6). However, levels of IL-6 correlated with the stage of disease; highest levels of IL-6 were found in LTBMC from patients with active myeloma. To identify the origin of IL-6 production, LTBMC from MM patients and normal donors were cocultured with BM-derived myeloma cells and cells from myeloma cell lines. IL-6 was induced by plasma cell lines that adhered to LTBMC such as ARH-77 and RPMI-8226, but not by nonadhering cell lines U266 and FRAVEL. Myeloma cells strongly stimulated IL-6 secretion in cocultures with LTBMC adherent cells from normal donors and myeloma patients. When direct cellular contact between LTBMC and plasma cells was prevented by tissue-culture inserts, no IL-6 production was induced. This implies that intimate cell-cell contact is a prerequisite for IL-6 induction. Binding of purified myeloma cells to LTBMC adherent cells was partly inhibited by monoclonal antibodies against adhesion molecules VLA-4, CD44, and lymphocyte function-associated antigen 1 (LFA-1) present on the plasma cell. Antibodies against VLA-4, CD29, and LFA-1 also inhibited the induced IL-6 secretion in plasma cell-LTBMC cocultures. In situ hybridization studies performed before and after coculture with plasma cells indicated that LTBMC adherent cells produce the IL-6. These results suggest that the high levels of IL-6 found in LTBMC of MM patients with active disease are a reflection of their previous contact with tumor cells in vivo. These results provide a new perspective on tumor growth in MM and emphasize the importance of plasma cell-LTBMC interaction in the pathophysiology of MM.
Nonrandom chromosomal rearrangements of 14q32.3 and 19p13.3 and preferential deletion of 1p in 21 patients with multiple myeloma and plasma cell leukemia
Authors: Taniwaki M , Nishida K , Takashima T , Nakagawa H , Fujii H , Tamaki T , Shimazaki C , Horiike S , Misawa S , Abe T
Published: October 2019

Structural chromosomal abnormalities and their break-points were characterized in 17 patients with multiple myeloma (MM) and 4 with plasma cell leukemia by banding. Chromosome 14q32 translocations with a variety of partners were detected in 13 patients, and a variant translocation t(8;22)(q24.1;q11) was detected in 1. Three recurrent 14q32 translocations have been identified: t(6;14)(p21.1;q32.3) occurring in 3 cases, and t(11;14)(q13;q32.3) and t(14;18) (q32.3;q21.3) each occurring in 2 cases. Translocations t(1;14)(q21;q32.3), t(3;14)(p11;q32),t(7;14)(q11.2;q32.3), and t(11;14)(q23;q32.3) were found in each patient, whereas in the remaining 2 patients, partner chromosomes could not be determined. The band 19p13.3 was newly delineated as a recurrent breakpoint involved in translocations in MM. Chromosomes 1 and 6 were also commonly involved in structural abnormalities (14 and 10 patients, respectively), although no particular bands were noted. However, the short arm of chromosome 1 was preferentially involved in deletion, suggesting a certain antioncogene on 1p associated with the development of myeloma. In addition; fluorescence in situ hybridization was successfully applied to determine the nature of the structural abnormalities in a patient with t(8;22) translocation. The present findings suggest that there may be subsets of 14q32 translocations specific to MM.
Expression and function of CD40 on Hodgkin and Reed-Sternberg cells and the possible relevance for Hodgkin's disease
Authors: Gruss HJ , Hirschstein D , Wright B , Ulrich D , Caligiuri MA , Barcos M , Strockbine L , Armitage RJ , Dower SK
Published: October 2019

CD40 was originally described as a B-cell-restricted antigen and was subsequently found to be a member of the tumor necrosis factor (TNF) receptor superfamily. CD40 is also expressed on dendritic cells, thymic epithelium, monocytes, and some carcinoma cell lines, and plays a critical role in cell contact-dependent activation. Primary and cultured Hodgkin and Reed-Sternberg (H-RS) cells, the presumed malignant cells of Hodgkin's disease (HD); were found to express high levels of cell surface CD40. We found that recombinant CD40 ligand (CD40L) induced interleukin-8 (IL-8) secretion and enhanced IL-6, TNF, and lymphotoxin-alpha (LT-alpha/TNF-beta) release from cultured H-RS cells. These cytokines play a significant role in the clinical presentation and pathology of HD, a tumor of cytokine-producing cells. CD40L had no mitogenic activity for HD-derived cell lines. In contrast, CD40L enhanced expression of costimulatory molecules intracellular adhesion molecule-T and B7–1 on cultured H-RS cells, both of which are overexpressed on primary H-RS cells. In addition, CD40L induced a 40% to 60% reduction of the expression of the HD-associated CD30 antigen, another member of the TNF receptor superfamily. Primary and cultured H- RS cells express not only CD30, but also CD40. CD40L has pleiotropic biologic activities on H-RS cells, and the CD40-CD40L interaction might be a critical element in the deregulated cytokine network and cell contact-dependent activation cascade typical for HD.
Structural requirements of platelet chemokines for neutrophil activation
Authors: Yan Z , Zhang J , Holt JC , Stewart GJ , Niewiarowski S , Poncz M
Published: October 2019

Using recombinantly expressed proteins and synthetic peptides, we examined the structural/functional features of the platelet chemokines, neutrophil-activating peptide-2 (NAP-2) and platelet factor 4 (PF4); that were important in their activation of neutrophils. Previous studies with the chemokine interleukin-8 (IL-8) had shown that the N- terminal region preceding the first cysteine residue was critical in defining neutrophil-activating properties. We examined whether NAP-2 and PF4 had similar structural requirements. In the Ale-glu-leu-arg (AELR) N-terminus of NAP-2, substitution of E or R abolished Ca2+ mobilization and elastase secretion. Unlike the parent molecule PF4, AELR/PF4, the hybrid formed by replacing the N-terminal sequence of PF4 before the first cysteine residue with the homologous sequence of NAP- 2, stimulated Ca2+ mobilization and elastase secretion. Furthermore, the effect of amino acid substitutions in the ELR motif differed from those seen with NAP-2 in that conserved substitutions of E or R in NAP- 2 abolished activity, but only reduced neutrophil activation in the hybrid. These studies show that just as with IL-8, the N-termini of NAP- 2 and PF4 are critical for high-level neutrophil-activating function. Desensitization studies provided information on receptor binding. NAP- 2, which binds almost exclusively to the type 2 IL-8 receptor (IL-8R), did not desensitize neutrophils to activation by IL-8 because IL-8 could bind to and activate via both type 1 and 2 IL-8R. AELR/PF4 appears to bind to both types of receptors because it desensitized neutrophils to NAP-2 activation; but was not desensitized by NAP-2, and because it desensitized to and was desensitized by IL-8. Thus, although NAP-2 and AELR/PF4 share approximately 60% amino acid homology, they have different receptor affinities. Studies were performed to define the role of the C-termini of these platelet chemokines in receptor binding. Heparin and a monoclonal antibody specific for the heparin- binding domain of PF4 both inhibited Ca2+ mobilization and elastase release, further suggesting that the C-terminus of these chemokines is important in receptor binding. Synthetic NAP-2(51–70) failed to mobilize Ca2+, whereas PF4(47-–70) and PF4(58–70) induced Ca2+ mobilization and secretion of elastase at high concentrations. Pertussis toxin inhibited neutrophil activation by 40% to 50%, establishing a role for G-protein-coupled receptors such as the IL-8Rs in activation by the PF4 C-terminal peptides.(ABSTRACT TRUNCATED AT 400 WORDS)