bluuebunny/crossref_metadata_2025 huggingface dataset:
Clonal karyotypic hematopoietic cell abnormalities occurring after autologous bone marrow transplantation for Hodgkin's disease and non- Hodgkin's lymphoma
Authors: Traweek ST , Slovak ML , Nademanee AP , Brynes RK , Niland JC , Forman SJ
Published: October 2019

Over a 6-year period, 275 patients were treated with autologous bone marrow transplantation (auto-BMT) for advanced-stage malignant lymphoma. After BMT, clonal chromosomal abnormalities were detected in hematopoietic cells from 10 patients. All 10 had morphologically and cytogenetically normal BMs at the time of stem cell harvest. The cytogenetic changes were first detected 1.8 to 6.5 years (mean, 3.9) after induction chemotherapy, and 0.5 to 3.1 years (mean, 1.4) after transplantation, and were characteristic of those reported for therapy- related myelodysplastic syndrome (MDS) in 9 of the patients: abnormalities of chromosome 5 or 7 (classical-form) were present in 4, 11q23 or 21q22 abnormalities (topoisomerase II-related form) were detected in 3, and a combination of both forms was seen in 2 patients. Clonal 2p abnormalities were found in the 1 remaining patient. The abnormal karyotypes were associated with morphologically recognizable MDS in 3 patients and with acute myeloid leukemia (AML) arising in MDS in 2. Four of these patients have died: 3 of AML and 1 of infection. One patient is still alive with cytopenia. The clonal cytogenetic abnormalities were not associated with MDS in 5 patients: 1 has died of recurrent lymphoma, 2 have cytopenia, and 2 still have no morphologic or clinical evidence of MDS after short follow-up (4 and 13 months). Compared with a control group matched for disease, length of follow-up, and treatment with auto-BMT, there were no statistically significant associations between the development of clonal chromosomal abnormalities and age, number of chemotherapeutic regimens, prior local radiation, BMT conditioning regimen (with or without total body irradiation), or type of lymphoma. These studies show that the risk of developing clonal cytogenetic changes after auto-BMT for malignant lymphoma is approximately 9% at 3 years, even when pre-BMT karyotypic studies are normal. The exact significance of these cytogenetic abnormalities in the absence of MDS or AML is unclear.
Differentiation-linked changes in tyrosine phosphorylation, functional activity, and gene expression downstream from the granulocyte- macrophage colony-stimulating factor receptor
Authors: Roberts PJ , Khwaja A , Lie AK , Bybee A , Yong K , Thomas NS , Linch DC
Published: October 2019

The HL-60 model of myeloid maturation was used to test whether changes in signaling from the granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor accompany maturation-related changes in cellular responses to GM-CSF. Receptor expression, tyrosine phosphorylation, functional activity, and c-fos gene expression were measured. Functional GM-CSF receptors were present throughout differentiation as both uninduced and dimethyl sulfoxide (DMSO)-induced HL-60 cells responded to GM-CSF, albeit in different ways. Uninduced promyelocytes proliferated in response to GM-CSF, whereas DMSO-induced cells lost the capacity to proliferate but did respond with increased expression of beta 2-integrins, enhanced respiratory burst activity, and metabolism of arachidonic acid. GM-CSF-stimulated upregulation of c-fos mRNA expression was not detected in immature cells but developed after 2 to 4 days with DMSO in line with a marked increase in responsiveness to stimulation with phorbol ester, showing that increased expression of c- fos is predominantly a feature of mature phagocytes. GM-CSF stimulated the tyrosine phosphorylation of a broadly similar range of proteins in both uninduced and DMSO-treated HL-60 cells, but protein bands were more heavily phosphorylated in DMSO-induced cells. Phosphorylation was rapid in onset and very transient in immature cells. Phosphorylation of several proteins, in particular a 130-kD band, was more sustained in DMSO-induced cells. These differences in signaling were not because of numerical differences in receptors, because reduction of GM-CSF concentration to trigger equivalent numbers of high-affinity receptors delayed the onset of phosphorylation in DMSO-induced cells. We conclude that there are maturation-related changes in signaling downstream from the GM-CSF receptor.
Clustering of vitronectin and RGD peptides on microspheres leads to engagement of integrins on the luminal aspect of endothelial cell membrane
Authors: Zanetti A , Conforti G , Hess S , Martin-Padura I , Ghibaudi E , Preissner KT , Dejana E
Published: October 2019

In previous work (Conforti et al, Blood 80:437, 1992), we have shown that integrins in endothelial cells (EC) are not polarized to the basal cell membrane, but are also exposed on the apical cell surface, in contact with blood. Therefore, endothelial integrins might be available for binding circulating plasma proteins. However soluble plasma vitronectin (vn) bound very poorly to EC apical surface and this interaction was unaffected by Arg-Gly-Asp (RGD) peptides or an anti- alpha v beta 3 serum. In contrast, beads (diameter, 4.5 microns) coupled with plasma vn associated to EC apical surface in a time- and concentration-dependent way. Addition of antibodies directed to vn, alpha v beta 3, and RGD-containing peptides blocked the interaction of vn beads with EC. In contrast, heparin and antibodies directed to alpha v beta 5 and beta 1 integrin chain had no effect. Beads coupled with Gly-Arg-Gly-Asp-Ser-Pro bound to the EC surface, but not those coupled with Gly-Arg-Gly-Glu-Ser-Pro. This interaction was blocked by alpha v beta 3 antibodies and RGD peptides, but not by alpha v beta 5 antibody. Overall, these results indicate that luminal alpha v beta 3 retains its binding capacity for surface-linked vn and RGD-containing ligands, but binding is observed only when the ligand is offered in a clustered, multivalent form. We propose that when vn or RGD-containing proteins are bound to circulating cells, they can act as bridging molecules by promoting adhesion of the cells to the endothelium via apical integrins.
Coagulation factor XII Locarno: the functional defect is caused by the amino acid substitution Arg 353-->Pro leading to loss of a kallikrein cleavage site
Authors: Hovinga JK , Schaller J , Stricker H , Wuillemin WA , Furlan M , Lammle B
Published: October 2019

The dysfunctional coagulation factor XII (FXII) Locarno was purified from 2 L of the proposita's plasma. Studies to identify the molecular defect responsible for the lack of amidolytic and proteolytic activity of this FXII variant were performed. Amino acid sequence analysis of peptides obtained from FXII Locarno on activation with either trypsin or plasma kallikrein and dextran sulfate showed an amino acid substitution of Arg 353 by Pro. Thereby, the kallikrein cleavage site at Arg 353-Val 354 is lost. Although trypsin-activated FXII Locarno was fully cleaved at Arg 334-Asn 335 and at Arg 343-Leu 344, neither amidolytic nor proteolytic activity was generated. We conclude that proteolytic cleavage at Arg 343 in the absence of cleavage at Arg 353 is not sufficient to expose the enzymatic active site in FXII Locarno.
Expression of EVI1 in myelodysplastic syndromes and other hematologic malignancies without 3q26 translocations
Authors: Russell M , List A , Greenberg P , Woodward S , Glinsmann B , Parganas E , Ihle J , Taetle R
Published: October 2019

The EVI1 gene encodes a zinc-finger, DNA-binding protein originally described as the transforming gene associated with a common ecotropic viral insertion site in myeloid leukemias. Previous studies demonstrated EVI1 expression in human leukemias in cases with 3q26 translocations, but not in normal blood or bone marrow. These studies also suggested an association between EVI1 expression and chromosome 7 deletion (del). Because of this association, we examined expression of EVI1 using RNA polymerase chain reaction (PCR) in patients with myelodysplastic syndromes (MDS) and acute leukemia with and without 3q26 translocations. EVI1 RNA was expressed in 29% of 34 (95% confidence interval, 20% to 50%) patients with the MDS subtypes refractory anemia (RA), refractory anemia with excess blasts (RAEB), or refractory anemia with excess blasts in transformation (RAEB-T). The vast majority of these cases occurred in patients with RAEB and RAEB-T. EVI1 expression was not detected in patients with chronic myelomonocytic leukemia (CMML), normal bone marrow or cord blood, or a variety of other hematologic malignancies. EVI1 RNA was detected in three of 18 patients with acute myelogenous leukemia (AML) and in two of four patients with acute promyelocytic leukemia (APL). Karyotypes showed that only one AML patient had karyotype 3q26 abnormalities, indicating that EVI1 expression is associated with cases that do not have structural abnormalities involving chromosome 3q26. These studies document for the first time the abnormal expression of EVI1 RNA by patients with MDS, and suggest an important role for EVI1 in the pathogenesis or progression of some myeloid malignancies.
Iron protoporphyrin IX (hemin) but not tin or zinc protoporphyrin IX can stimulate gene expression in K562 cells from enhancer elements containing binding sites for NF-E2
Authors: Palma JF , Gao X , Lin CH , Wu S , Solomon WB
Published: October 2019

Many genes whose transcription is erythroid-specific contain enhancer or promoter elements that bind the transcription factor NF-E2. Hemin induction increases the expression of globin genes in the human erythroleukemia cell line K562, and increases the expression of reporters gene regulated by an enhancer elements containing binding sites for NF-E2. The failure of metalloporphyrins other than hemin to stimulate the transient expression of a CAT reporter gene linked to an enhancer element containing a binding site for NF-E2 was correlated with their failure to induce benzidine-positive K562 cells and increase the steady-state level of gamma-globin mRNA. This study suggests that elevated levels of zinc protoporphyrin IX found in the anemia of chronic disease, iron deficiency, and lead poisoning may contribute to a decrease in globin gene expression by interfering with the transcriptional activity of enhancer elements containing binding sites for NF-E2.
Transfer and expression of the human multiple drug resistance gene in human CD34+ cells
Authors: Ward M , Richardson C , Pioli P , Smith L , Podda S , Goff S , Hesdorffer C , Bank A
Published: October 2019

The human multiple-drug resistance (MDR1) gene has been transferred into human hematopoietic progenitors using retroviral gene transfer. Human bone marrow cells and isolated CD34+ cells isolated from marrow were exposed to growth factors interleukin-3 (IL-3), IL-6, and stem cell factor for 48 hours and then to two changes of MDR retroviral supernatants over the next 24 hours. Progenitor assays in methylcellulose at this time showed that 18% to 70% of BFU-E and 30% to 60% of CFU-GM contain the transferred MDR gene by polymerase chain reaction analysis. Up to 11.2% of the progeny of these cells express increased amounts of MDR glycoprotein on their surface by fluorescence- activated cell sorter (FACS) analysis. In addition, transduced cells are enriched in high MDR-expressing cells after exposure to taxol as assessed by FACS analysis, and by resistance of BFU-E to taxol (Bristol- Myers Squibb, Princeton, NJ). These studies indicate the feasibility of using MDR gene transfer as a means of enriching marrow for MDR- transduced cells. They also provide the basis of a phase 1 clinical protocol in patients with advanced cancers not involving the bone marrow for the use of MDR gene transfer as a means of protecting marrow cells, which normally express low levels of MDR, from the myelosuppressive effects of drugs like taxol.
Annexin V for flow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis
Authors: Koopman G , Reutelingsperger CP , Kuijten GA , Keehnen RM , Pals ST , van Oers MH
Published: October 2019

Apoptosis, or programmed cell death, is a general mechanism for removal of unwanted cells from the immune system. It is characterized by chromatin condensation, a reduction in cell volume, and endonuclease cleavage of DNA into oligonucleosomal length fragments. Apoptosis is also accompanied by a loss of membrane phospholipid asymmetry, resulting in the exposure of phosphatidylserine at the surface of the cell. Expression of phosphatidylserine at the cell surface plays an important role in the recognition and removal of apoptotic cells by macrophages. Here we describe a new method for the detection of apoptotic cells by flow cytometry, using the binding of fluorescein isothiocyanate-labeled annexin V to phosphatidylserine. When Burkitt lymphoma cell lines and freshly isolated germinal center B cells are cultured under apoptosis inducing conditions, all cells showing chromatin condensation strongly stain with annexin V, whereas normal cells are annexin V negative. Moreover, DNA fragmentation is only found in the annexin V-positive cells. The nonvital dye ethidium bromide was found to stain a subpopulation of the annexin V-positive apoptotic cells, increasing with time. Our results indicate that the phase in apoptosis that is characterized by chromatin condensation coincides with phosphatidylserine exposure. Importantly, it precedes membrane damage that might lead to release from the cells of enzymes that are harmful to the surrounding tissues. Annexin V may prove important in further unravelling the regulation of apoptosis.
The tetrapeptide AcSDKP specifically blocks the cycling of primitive normal but not leukemic progenitors in long-term culture: evidence for an indirect mechanism
Authors: Cashman JD , Eaves AC , Eaves CJ
Published: October 2019

In the present study, we investigated the ability of the tetrapeptide NAc-Ser-Asp-Lys-Pro-OH (AcSDKP), a reported inhibitor of primitive hematopoietic cells, to influence the proliferative behavior of primitive normal and chronic myeloid leukemia (CML) progenitor cells in the adherent layer of long-term cultures (LTCs). Addition of > or = 50 ng/mL of AcSDKP to LTCs of normal cells at the time of the regular weekly half-medium change selectively and reversibly decreased the proportion of high proliferative potential erythroid and granulopoietic progenitors in the adherent layer that were in S-phase without changing their numbers, but had no effect on either the cycling activity or number of analogous (neoplastic) cells in the adherent layer of CML LTCs. Specificity of the effect of AcSDKP on primitive normal progenitors was demonstrated by the finding that a similar addition of either the control peptide, AcSDKE, or 100 ng/mL of tumor necrosis factor-alpha (TNF-alpha, which contains the SDKP sequence), or SDKP itself (at 300 ng/mL) did not inhibit the proliferation of primitive normal progenitors in LTC adherent layers. Incorporation of > or = 30 ng/mL of AcSDKP (but not the related control peptide, AcSDKE) directly into methylcellulose cultures of normal marrow cells resulted in a dose- dependent suppression of colony formation, which was not seen in similar studies with CML marrow or after removal of adherent cells from normal marrow. Additional experiments showed that the inhibitory effect of AcSDKP on primitive normal progenitor cycling in the LTC system could be overcome by the simultaneous addition of macrophage inflammatory protein-1 beta (MIP-1 beta); an antagonist of MIP-1 alpha. The apparent differential effect of AcSDKP on primitive normal and CML progenitors may thus be a secondary consequence of the differential responsiveness of these cells to MIP-1 alpha for another molecule antagonized by MIP-1 beta), whose production or release by adherent marrow cells is inducible by AcSDKP. Such a mechanism may offer a method for obtaining localized increases in vivo of cytokines like MIP- 1 alpha, suggesting novel and perhaps less toxic strategies for protecting primitive normal progenitors during repeated treatments with cycle-active chemotherapeutic agents where escalating the dose of drug given would be desirable.
Activation of phosphatidylinositol-3 kinase by ligation of the interleukin-7 receptor is dependent on protein tyrosine kinase activity
Authors: Dadi H , Ke S , Roifman CM
Published: October 2019

Ligation of the interleukin-7 receptor (IL-7R) results in a rapid phosphorylation of tyrosine residues on multiple substrates. In addition, we have recently shown that the IL-7R mediates activation of phosphatidylinositol-3 (PI-3) kinase. Because PI-3 kinase activity can be immunoprecipitated with antiphosphotyrosine antibodies in most receptor systems studied, it has been examined that either PI-3 kinase or an associated protein become tyrosine-phosphorylated after ligand binding. We studied here the possibility that PI-3 kinase, which is directly linked to mitogenic responses in growth factor receptors, is tyrosine-phosphorylated after stimulation of the IL-7R. Using anti-p85 alpha or anti-p85 beta antibodies raised against the p85 subunit of PI- 3 kinase for immunoprecipitation and subsequent blotting with antiphosphotyrosine clearly shows that IL-7-stimulated human precursor cells contain both p85 alpha and p85 beta proteins phosphorylated on tyrosine residues. Specific protein tyrosine kinase inhibitors such as tyrphostin AG-490 block total cell lysate phosphorylation and tyrosine phosphorylation on p85. Similar concentrations of this inhibitor also block in vitro and in vivo PI-3 kinase activity suggesting that this enzyme activation is dependent on the phosphorylation event of p85. In addition, AG-490 blocks IL-7-mediated proliferation in a dose-dependent manner, suggesting a link between the early events of PI-3 kinase phosphorylation and activation with IL-7R-induced cell growth.
BCL-2 expression and mitochondrial activity in leukemic cells with different sensitivity to glucocorticoid-induced apoptosis
Authors: Smets LA , Van den Berg J , Acton D , Top B , Van Rooij H , Verwijs-Janssen M
Published: October 2019

The present study investigates the relationship between mitochondrial activity and the expression of the BCL-2 gene in a panel of six human and murine leukemia/lymphoma cell lines. The cell lines all contained normal glucocorticoid receptors but differed widely in sensitivity to dexamethasone, ranging from very sensitive S49 lymphoma to completely resistant HL-60 acute leukemia cells. In this panel, 10- to 15-fold differences in basal adenosine triphosphate (ATP) content and adenosine diphosphate (ADP)/ATP ratio were correlated with up to fivefold differences in bcl-2 protein (in human cells) and approximately 25-fold difference in bcl-2 mRNA content (all cell lines). Moreover, ATP content and BCL-2 gene expression were inversely correlated with glucocorticoid sensitivity and cell cycle length. In resistant cell lines, sensitivity to dexamethasone was restored by the mitochondrial inhibitors rotenone and meta-iodobenzylguanidine. This sensitization was not accompanied by detectable reductions in bcl-2 mRNA or protein content, suggesting that the inhibitors were capable of overriding BCL- 2-mediated inhibition of apoptosis. Increased mitochondrial activity and (overexpressed) BCL-2 appeared closely related properties of glucocorticoid-resistant cells, sharing common cellular targets in hormone-induced apoptosis.
Clinical spectrum of clonal proliferations of T-large granular lymphocytes: a T-cell clonopathy of undetermined significance?
Authors: Dhodapkar MV , Li CY , Lust JA , Tefferi A , Phyliky RL
Published: October 2019

We identified 68 patients with clonal T-large granular lymphocyte (T- LGL) proliferations who were seen at the Mayo Clinic between 1984 and 1992. Nineteen (28%) were asymptomatic at diagnosis, while the rest experienced fatigue (60%), B-symptoms (12%), and recurrent infections (15%). Associated comorbid conditions included rheumatoid arthritis (RA) in 26%. Severe anemia (hemoglobin [Hb] < 8g/dL) and neutopenia (absolute neutrophil count [ANC] < 500/microL) were seen in 19% and 40% of patients, respectively. Immunophenotypic studies showed CD3+, CD8+ phenotype in the majority (72%). Twenty-one patients (31%) have required no therapy, and remain relatively stable with a median follow- up period of 50 months. Treatment was required at either diagnosis (36 patients) or at subsequent follow-up (11 patients). Initial response rates were similar in patients treated with cyclophosphamide (CTX) with or without prednisone (69%), or prednisone alone (73%). Overall, 61 patients (90%) are alive with a median follow-up of 44 months. Actuarial median survival of this entire cohort is 161 months. The presence of anemia or symptoms does not appear to correlate with the tumor burden. In patients requiring therapy, a lower ANC and the presence of B-symptoms/infection were independently associated with a significantly lower probability of achieving a molecular or hematologic complete remission (H-CR). Intermittent immunosuppressive therapy is effective in achieving durable responses in a number of patients. T-LGL proliferations are associated with a favorable prognosis and response to therapy. However, significant heterogeneity exists in clinical presentation and associated comorbid conditions. These disorders should be included in the differential diagnosis of patients with unexplained cytopenias, particularly in the setting of RA and other autoimmune disorders. Analogous to the situation with monoclonal gammopathies, a term such as T-cell clonopathy of undetermined significance (TCUS) may be more appropriate to describe these patients.
Hematologic abnormalities in Fanconi anemia: an International Fanconi Anemia Registry study [see comments]
Authors: Butturini A , Gale RP , Verlander PC , Adler-Brecher B , Gillio AP , Auerbach AD
Published: October 2019

We analyzed data from 388 subjects with Fanconi anemia reported to the International Fanconi Anemia Registry (IFAR). Of those, 332 developed hematologic abnormalities at a median age of 7 years (range, birth to 31 years). Actuarial risk of developing hematopoietic abnormalities was 98% (95% confidence interval, 93% to 99%) by 40 years of age. Common hematologic abnormalities were thrombocytopenia and pancytopenia. These were often associated with decreased bone marrow (BM) cellularity (75% of cases studied). Clonal cytogenetic abnormalities developed in 23 of 68 persons with BM failure who had adequate studies. Actuarial risk of clonal cytogenetic abnormalities during BM failure was 67% (47% to 87%) by 30 years of age. Fifty-nine subjects developed myelodysplastic syndrome (MDS) or acute myelogenous leukemia (AML). Actuarial risk of MDS or AML was 52% (37% to 67%) by 40 years of age. Risk was higher in persons with than in those without a prior clonal cytogenetic abnormality (3% [0% to 9%] v 35% [0% to 79%]; P = .006). One hundred twenty persons died of hematologic causes including BM failure, MDS or AML and treatment related complications. Actuarial risk of death from hematologic causes was 81% (67% to 90%) by 40 years of age.
Mice lacking granulocyte colony-stimulating factor have chronic neutropenia, granulocyte and macrophage progenitor cell deficiency, and impaired neutrophil mobilization
Authors: Lieschke GJ , Grail D , Hodgson G , Metcalf D , Stanley E , Cheers C , Fowler KJ , Basu S , Zhan YF , Dunn AR
Published: October 2019

Mice lacking granulocyte colony-stimulating factor (G-CSF) were generated by targeted disruption of the G-CSF gene in embryonal stem cells. G-CSF-deficient mice (genotype G-CSF-/-) are viable, fertile, and superficially healthy, but have a chronic neutropenia. Peripheral blood neutrophil levels were 20% to 30% of wild-type mice (genotype G- CSF+/+) and mice heterozygous for the null mutation had intermediate neutrophil levels, suggesting a gene-dosage effect. In the marrow of G- CSF-/- mice, granulopoietic precursor cells were reduced by 50% and there were reduced levels of granulocyte, macrophage, and blast progenitor cells. Despite G-CSF deficiency, mature neutrophils were still present in the blood and marrow, indicating that other factors can support neutrophil production in vivo. G-CSF-/- mice had reduced numbers of neutrophils available for rapid mobilization into the circulation by a single dose of G-CSF. G-CSF administration reversed the granulopoietic defect of G-CSF-/- mice. One day of G-CSF administration to G-CSF-/- mice elevated circulating neutrophil levels to normal, and after 4 days of G-CSF administration, G-CSF+/+ and G-CSF- /- marrows were morphologically indistinguishable. G-CSF-/- mice had a markedly impaired ability to control infection with Listeria monocytogenes, with diminished neutrophil and delayed monocyte increases in the blood and reduced infection-driven granulopoiesis. Collectively, these observations indicate that G-CSF is indispensible for maintaining the normal quantitative balance of neutrophil production during “steady-state” granulopoiesis in vivo and also implicate G-CSF in “emergency” granulopoiesis during infections.
Substitutions and deletions in the cytoplasmic domain of the phagocytic receptor Fc gamma RIIA: effect on receptor tyrosine phosphorylation and phagocytosis [published erratum appears in Blood 1994 Nov 1;84(9):3252]
Authors: Mitchell MA , Huang MM , Chien P , Indik ZK , Pan XQ , Schreiber AD
Published: October 2019

Fc gamma RIIA in the absence of other Fc receptors or receptor subunits induces the ingestion of IgG-coated cells. The cytoplasmic domain of Fc gamma RIIA contains two Y-x-x-L sequences similar to those in other Ig gene family receptors plus an additional tyrosine residue not in a Y-x- x-L motif. Upon cross-linking, Fc gamma RIIA is phosphorylated on tyrosine and the cytoplasmic tyrosines, Y275 (Y1), Y282 (Y2), and Y298 (Y3), may be important for its phagocytic activity. Because COS-1 cells can serve as a model for examining molecular structures involved in phagocytosis, substitutions and deletions were introduced into the cytoplasmic domain of Fc gamma RIIA and examined in COS-1 cell transfectants for their effects on phagocytosis and tyrosine phosphorylation. Disruption of a single cytoplasmic Y-x-x-L motif by substitution of tyrosine Y2 or Y3 by phenylalanine or by removing the threonine and leucine residues within the motif inhibited phagocytosis 50% to 65%. Tyrosine phosphorylation of Fc gamma RIIA also was inhibited, although to a greater extent by the substitution of Y3 than of Y2. Replacement of the N-terminal first cytoplasmic domain tyrosine, Y1, which is not within a typical Y-x-x-L, by itself did not inhibit phagocytosis, but replacement of Y1 in mutants lacking Y2 or Y3 virtually eliminated phagocytic activity and receptor tyrosine phosphorylation. Thus, at least two cytoplasmic tyrosines, including at least one typical single Y-x-x-L motif, are required for phagocytosis by Fc gamma RIIA. The data suggest that there is a close but not a simple relationship between phosphorylation of the Fc gamma RIIA cytoplasmic tyrosines and Fc gamma RIIA-mediated phagocytosis. Y3 appears to be particularly important because its removal by truncation or replacement with phenylalanine inhibits both tyrosine phosphorylation and phagocytosis in parallel. Alterations in the 12 residue proline-containing sequence between the two Y-x-x-L motifs also reduced phagocytic activity and tyrosine phosphorylation. Thus, the specific structure of the Fc gamma RIIA cytoplasmic domain accounts for its ability to stimulate phagocytosis in the absence of other subunits.
Accelerated healing of chronic sickle-cell leg ulcers treated with RGD peptide matrix. RGD Study Group
Authors: Wethers DL , Ramirez GM , Koshy M , Steinberg MH , Phillips G Jr , Siegel RS , Eckman JR , Prchal JT
Published: October 2019

Leg ulcers are a chronic manifestation of sickle-cell disease (SCD) and are often painful, disabling, and difficult to treat. RGD peptide matrix treatment is a novel therapy designed to provide a topical synthetic extracellular matrix that can act as a temporary substitute for the damaged natural matrix at the ulcer site. In this randomized, placebo-controlled, double-blind, prospective, multicenter investigation, SCD patients with full-thickness leg ulcers were treated with standard therapy plus RGD peptide matrix or saline placebo once weekly for up to 10 weeks. Healing in patients with chronic ulcers (2 months or greater in duration) was significantly accelerated (P = .0085) in RGD peptide matrix recipients compared with the placebo group. In these chronic ulcer cases, the average percent ulcer closure (decrease in ulcer surface area) in the RGD peptide matrix group (54.4% +/- 8.9%) exceeded that in the placebo group (19.0% +/- 24.3%) nearly threefold by study endpoint. Furthermore, RGD peptide matrix was equally effective in promoting healing of long persistent ulcers and ulcers of shorter duration. In contrast, standard therapy plus placebo was significantly less effective (P = .001) in promoting healing for ulcers of progressively greater duration. The results of this study provide preliminary evidence that RGD peptide matrix treatment may significantly accelerate healing of chronic sickle-cell leg ulcers.
The active monomeric form of macrophage inflammatory protein-1 alpha interacts with high- and low-affinity classes of receptors on human hematopoietic cells
Authors: Avalos BR , Bartynski KJ , Elder PJ , Kotur MS , Burton WG , Wilkie NM
Published: October 2019

Macrophage inflammatory protein-1 alpha (MIP-1 alpha) and its human homologue GOS19.1/LD78 are members of the C-C chemokine/intercrine family of secreted proteins. They have proinflammatory properties and also inhibit cell cycle progression of hematopoietic stem cells. Characterization of MIP-1 alpha receptor(s) has been confused because of its reported aggregation to inactive forms. Using a defined monomeric form of MIP-1 alpha that is biologically active for stem cell inhibition and induction of oxidative metabolism in polymorphonuclear cells, we report the detection of high- and low-affinity receptor classes on human leukemic CD34+ blast cells, promyelocytic cells, monocytes, peripheral blood neutrophils, and T cells. Both high- and low-affinity classes are expressed simultaneously in promyelocytes and neutrophils. The calculated kd for high-affinity receptors correlates with the concentrations of MIP-1 alpha required to induce a biologic effect on stem cells and neutrophils. Cross-linking studies show that MIP-1 alpha associates with two cell surface proteins with apparent molecular masses of 92 kD and 52 kD. Direct competition binding studies combined with studies on the inhibition of stem cells show that human and murine MIP-1 alpha have different receptor-binding and biologic properties.
An autoantibody directed against human thrombin anion-binding exosite in a patient with arterial thrombosis: effects on platelets, endothelial cells, and protein C activation
Authors: Arnaud E , Lafay M , Gaussem P , Picard V , Jandrot-Perrus M , Aiach M , Rendu F
Published: October 2019

An autoantibody, developed by a patient with severe and recurrent arterial thrombosis, was characterized to be directed against the anion- binding exosite of thrombin, and inhibited all thrombin interactions requiring this secondary binding site without interfering with the catalytic site. The effect of the antibody was studied on thrombin interactions with platelets and endothelial cells from human umbilical veins (HUVEC). The autoantibody specifically and concentration- dependently inhibited alpha-thrombin-induced platelet activation and prostacyclin (PGI2) synthesis from HUVEC. It had no effect when gamma- thrombin or the thrombin receptor activation peptide SFLLR were the inducers. The effect of the antibody on protein C activation has been studied. The antibody blocked the thrombin-thrombomodulin activation of protein C. The inhibition of the activation was maximal with a low concentration of thrombomodulin. The fact that the autoantibody inhibited concentration-dependent alpha-thrombin-induced platelet and endothelial cell functions emphasizes the crucial role of the anion- binding exosite of thrombin to activate its receptor. In regard to the pathology, the antibody inhibited two vascular processes implicated in thrombin-antithrombotic functions, PGI2 secretion, and protein C activation, which could be implicated in this arterial thrombotic disease.
A new substitution, gamma 358 Ser-->Cys, in fibrinogen Milano VII causes defective fibrin polymerization
Authors: Steinmann C , Bogli C , Jungo M , Lammle B , Heinemann G , Wermuth B , Redaelli R , Baudo F , Furlan M
Published: October 2019

Fibrinogen Milano VII is a hereditary fibrinogen variant detected in a woman with no clinical symptoms of bleeding or thrombosis. Thrombin and reptilase clotting times were prolonged in six family members from three generations. Release of fibrinopeptides A and B was normal. Fibrin polymerization was strongly delayed both in the presence and in the absence of calcium. The structural defect was determined by sequence analysis of a 290-bp fragment of genomic DNA amplified by polymerase chain reaction and cloned in M13mp19. The triplet TCT coding for the amino acid residue gamma 358 was found to be replaced by TGT, resulting in the substitution gamma 358 Ser-->Cys. Immunoblot analysis demonstrated the presence of covalently linked fibrinogen albumin and fibrinogen (albumin)2 complexes. Albumin was released from fibrinogen Milano VII by limited reduction with 2-mercaptoethanol. Fibrin polymerization was not normalized after removal of albumin from fibrinogen Milano VII, suggesting that the delayed clot formation is not due to steric hindrance caused by bound albumin but by substitution of gamma 358 Ser by Cys itself. Our results indicate that the residue gamma 358 Ser is essential for normal expression of the carboxy terminal polymerization site on the fibrinogen gamma-chain.
Granulocyte colony-stimulating factor (CSF) but not interleukin-1 (IL- 1), IL-3, and granulocyte-macrophage CSF protect bone marrow progenitor cells from suppression by allosensitized cytotoxic T cells
Authors: Gerritsen WR , O'Reilly RJ
Published: October 2019

The major immunological reactions after an allogeneic bone marrow transplantation (BMT) are graft rejection and graft-versus-host disease (GVHD). GVHD can be prevented by T-cell depletion of the allogeneic BM graft, but the beneficial effect of T-cell depletion on the incidence of GVHD is counterbalanced by a higher incidence of graft failure. One option for the prevention of graft rejection after T-cell-depleted BM grafts is the administration of cytokines. Before applying cytokines after an allogeneic BMT, we considered it desirable to learn whether cytokines would alter the susceptibility of donor BM cells to host T cells. An in vitro assay was developed to investigate the role of the cytokines interleukin-1 (IL-1), IL-3, granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage CSF (GM-CSF) on the interaction between allosensitized, cytotoxic-T cells (CTLs) and T-cell- depleted BM cells. CTLs primed against the BM donor suppressed the formation of colonies consisting of granulocytes and macrophages (colony-forming unit GM). Colony formation was not inhibited by CTLs sensitized against a third party. Accordingly, the number of colonies scored in cocultures with CTLs sensitized to third party antigens were designated as 0% inhibition. A 66% inhibition of colony formation was observed for untreated BM cells at an effector:target (E:T) ratio of 1:1. Pretreatment of the BM cells with the cytokines G-CSF, GM-CSF, IL- 1, and IL-3 resulted in a 38% (P = .001), 53%, 66%, and 68% inhibition of colony formation, respectively, at E:T ratios of 1:1. G-CSF reduced the susceptibility of BM cells over a range from 4:1 to 1:16 (E:T ratios). GM-CSF had only significant influence at the lower E:T ratios (1:4 and 1:16). These in vitro data indicate that G-CSF could protect BM cells from killing by allosensitized CTLs and suggest that administration of these cytokines might potentially reduce the susceptibility of T-cell-depleted allogeneic BM grafts to host T-cell- mediated rejection.
Expression of axl, a transforming receptor tyrosine kinase, in normal and malignant hematopoiesis
Authors: Neubauer A , Fiebeler A , Graham DK , O'Bryan JP , Schmidt CA , Barckow P , Serke S , Siegert W , Snodgrass HR , Huhn D
Published: October 2019

We previously reported the cloning, and characterization of a receptor tyrosine kinase, axl, from two patients with chronic myelogenous leukemia. Herein, we describe the expression pattern of axl in normal and malignant hematopoietic tissue axl message is detected in normal human bone marrow but not significantly in normal blood leukocytes. Cell separation experiments showed that axl is expressed in hematopoietic CD34+ progenitor and marrow stromal cells, at low levels in peripheral monocytes, but not in lymphocytes or granulocytes. Consistent with the normal pattern of axl expression, axl RNA was found predominantly in diseases of the myeloid lineage: 39 of 66 (59%) patients with myeloproliferative disorders (acute myeloid leukemia, chronic myeloid leukemia (CML) in chronic phase, CML in myeloid blast crisis, and myelodysplasia) showed significant axl transcription, as compared with 1 of 45 (2%) lymphoid leukemias (chronic lymphocytic leukemia, acute lymphocytic leukemia, and CML in lymphoid blast crisis). Treatment of K562 cells with the phorbol ester, 12-O- tetradecanoylphorbol-13-acetate (TPA), administration of interferon alpha (IFN alpha) to normal monocytes, and treatment of U937 cells with TPA and IFN tau significantly induced axl expression, supporting a role for this kinase in the intracellular signaling of myeloid cells through a variety of biochemical pathways. These results suggest that the axl kinase may be operative in normal and malignant myeloid biology.
Primary leukemia cells resistant to alpha-interferon in vitro are defective in the activation of the DNA-binding factor interferon- stimulated gene factor 3
Authors: Xu B , Grander D , Sangfelt O , Einhorn S
Published: October 2019

Cells from one-third of chronic lymphocytic leukemia (CLL) patients are resistant to alpha-interferon (alpha-IFN) as measured by induction of blast transformation. We have previously shown that all CLL clones express alpha/beta-IFN receptors, but that the resistant cells are defective in the induction of the enzyme 2′,5′-oligoadenylate synthetase (2′,5-A synthetase). Thus, the deficiency in IFN sensitivity is localized somewhere between the interaction of the IFN molecule with its receptor and induction of 2′,5′-A synthetase. We have now further characterized the resistance of CLL clones to IFN by investigating whether it is associated with a defect in the activation of IFN- stimulated gene factor 3 (ISGF3), which is involved in the activation of alpha-IFN-stimulated genes (ISGs). A defect induction of ISGF3 after alpha-IFN treatment was found in 4 of 12 CLL patients. There was a close correlation between defective induction of ISGF3 and a lack of enhancement of 2′,5′-A synthetase as well as induction of blast transformation. Pretreatment with gamma-IFN and mixing experiments with extracts from IFN-sensitive cells indicate that a lack of the gamma- component of ISGF3 was the reason for defect in activation in 2 of the patients. We conclude that a defect in activation of ISGF3 is a possible cause for resistance in CLL cells to IFN-induced blast transformation in vitro.
Anti-erythropoietin (EPO) receptor monoclonal antibodies distinguish EPO-dependent and EPO-independent erythroid progenitors in polycythemia vera
Authors: Fisher MJ , Prchal JF , Prchal JT , D'Andrea AD
Published: October 2019

Erythroid progenitor cells isolated from patients with polycythemia vera (PV) proliferate and differentiate in methylcellulose in the absence of exogenous erythropoietin (EPO). To investigate the potential role of the erythropoietin receptor (EPO-R) in the pathogenesis of PV, we cultured bone marrow-derived or peripheral blood-derived erythroid progenitors in the presence of neutralizing monoclonal antibodies (MoAbs) specific for EPO or EPO-R. Mononuclear cells were obtained from 9 healthy adults and 9 PV patients by Ficoll-Hypaque gradients and cultured with or without EPO in methylcellulose for 12 days under standard or serum-free conditions. Neutralizing anti-EPO and anti-EPO-R MoAbs, added to cultures at day 0, caused dose-dependent growth inhibition of all normal burst-forming units-erythroid (BFU-E) derived from health adult controls. The MoAbs had no effect on the growth of nonerythroid progenitor cells under the same culture conditions. In contrast, neutralizing antibodies distinguished two classes of BFU-E derived from PV patients. Class I BFU-E from PV patients were EPO- dependent. These progenitors, like those derived from healthy adults, had normal EPO dose-dependent growth characteristics and showed a normal period of EPO requirement in vitro that extended 6 days after the initiation of culture. These results indicate that EPO exerts its critical effect early during erythroid differentiation; the addition of neutralizing antibodies to normal progenitors after 6 days had no effect on the subsequent size or maturation of the colonies. Class II BFU-E from PV patients were EPO-independent. They proliferated and differentiated even in the presence of high concentrations of neutralizing anti-EPO or anti-EPO-R MoAbs. We conclude that the class II BFU-E from PV patients are independent of free EPO.
The structure of the human red blood cell anion exchanger (EPB3, AE1, band 3) gene
Authors: Schofield AE , Martin PG , Spillett D , Tanner MJ
Published: October 2019

The structure and sequence of the human red blood cell anion exchanger (EPB3, AE1, band 3) gene was determined by analysis of genomic and cDNA clones. The gene extends over 18 kb and consists of 20 exons. The cDNA sequence comprises 4,906 nucleotides [excluding the poly(A) tail]. There is extensive similarity between the human and mouse AE1 gene, although the latter covers 17 kb. The additional sequence present within the human AE1 gene compared with the mouse gene is mainly caused by the presence of 6 Alu repetitive units in the human gene between intron 13 and exon 20, one of which is within the 3′ noncoding region of the cDNA. Two potential promoter regions within the human AE1 gene are located so that they could give rise to the different transcripts found in erythroid cells and in the kidney. The kidney transcript would lack exons 1 through 3 of the erythroid transcript. The translation initiator downstream to the human kidney promoter would give rise to a protein with a 20 amino acid section at the N-terminus that is not present in the erythroid human AE1 protein or the rodent kidney AE1 proteins.
Interleukin-1 alpha administered after autologous transplantation: a phase I/II clinical trial
Authors: Weisdorf D , Katsanis E , Verfaillie C , Ramsay NK , Haake R , Garrison L , Blazar BR
Published: October 2019

Interleukin-1 alpha (IL-1 alpha) can act as both a hematopoietic growth factor and a stimulant of cellular and humoral immune responses. To promote acceleration of hematologic recovery and induce immune antitumor activity, we initiated a phase I/II dose escalation trial of 6-hour daily infusions of recombinant human IL-1 alpha after autologous transplantation. Forty patients with Hodgkin's disease (n = 9) and non- Hodgkin's lymphoma (n = 31) transplanted with unmobilized autologous peripheral blood stem cells or bone marrow stem cells received daily 6- hour infusions of IL-1 alpha (day 0 to day +13) at daily doses between 0.1 to 10 micrograms/m2/d; 7 patients received only 7 planned days of IL-1 alpha (day 0 through 6). Most patients received all 14 days of therapy, although 5 patients discontinued treatment early (after 1 to 6 doses) because of fever and severe chills. Toxicity included IL-1 alpha- related fever (occurring on a median of 9 of 14 treatment days), fatigue, and severe chills. Hypotension was dose-limiting and led to discontinuation of IL-1 alpha in both patients receiving 10 micrograms/m2/d. IL-1 alpha-treated patients receiving 3.0 micrograms/m2/d (the maximum tolerated dose) achieved neutrophil recovery (absolute neutrophil count greater than 500/microL) significantly earlier (median, 12 days; range, 11 to 27) than untreated control patients or those receiving IL-1 alpha at 0.1 to 1.0 micrograms/m2/d (median, 27; range, 9 to 63; P < .0001). In addition, the IL-1 alpha patients' bone marrows at day +14 were significantly enriched with committed myeloid progenitor cells. Strong trends to earlier freedom from red blood cell (P = .06) and platelet (P = .09) transfusions were also noted after IL-1 alpha treatment. This earlier hematopoietic engraftment after 3.0 micrograms/m2/d IL-1 alpha allowed earlier hospital discharge (median, 25 v 37 days for control or low- dose IL-1 alpha patients [P < .0001]) and a concomitant reduction (by $38,000) in median hospital charges (P = .01). The clinical toxicities of IL-1 alpha infusion are substantial, though not life-threatening. The accelerated hematopoiesis and immune response activation observed in this trial suggest the value of its further investigation in controlled trials and perhaps in combination with other hemopoietins after transplantation.
Graft-versus-host disease: new directions for a persistent problem
Authors: Vogelsang GB , Hess AD
Published: October 2019

Graft-versus-host disease (GVHD) continues to be a major complication after allogeneic bone marrow transplantation, especially with the increasing use of unrelated and mismatched donors. Recently there has been renewed scientific interest in GVHD because of the increasing appreciation of the complexity of the immune responses seen in GVHD. Two basic aspects of the immune response in GVHD, the immunologic target and the effector mechanisms, are now more completely understood. First, the target of the immune response in GVHD has long been felt to be histocompatibility antigens possessed by the host, but not the donor. Recently, recognition of self antigens in GVHD has been documented, showing that GVHD is more complex than simple alloreactivity. Second, the effector mechanism in GVHD was initially felt to be direct cytotoxicity by alloreactive T cells. It is now recognized that cytokines play a central role in mediating many of the clinical and experimental manifestations of GVHD. The development in these two areas will be reviewed and the implications for clinical transplantation discussed.
Busulfan bioavailability
Authors: Hassan M , Ljungman P , Bolme P , Ringden O , Syruckova Z , Bekassy A , Stary J , Wallin I , Kallberg N
Published: October 2019

Busulfan is widely used as a component of the myeloablative therapy in bone marrow transplantation. Recent studies have shown that the drug disposition is altered in children and is associated with less therapeutic effectiveness, lower toxicities, and higher rates of engraftment failure. We have evaluated the bioavailability of the drug in two groups of patients: eight children between 1.5 and 6 years of age and eight older children and adults between 13 and 60 years. Oral bioavailability showed a large interindividual variation. In children, the bioavailability ranged from 0.22 to 1.20, and for adults, it was within the range 0.47 to 1.03. The elimination half-life after intravenous administration in children (2.46 +/- 0.27 hours; mean +/- SD) did not differ from that obtained for adults (2.61 +/- 0.62 hours). However, busulfan clearance normalized to body weight was significantly higher in children (3.62 +/- 0.78 mL.min-1.kg-1) than that in adults (2.49 +/- 0.52 mL.min-1.kg-1). Also, the distribution volume normalized for body weight was significantly higher in children (0.74 +/- 0.10 L.kg-1) compared with 0.56 +/- 0.10 L. kg-1 in adults. The difference in clearance between children and adults was not statistically significant when normalized to body surface area, which most probably shows that busulfan dosage should be calculated on the basis of surface area rather than body weight. However, to avoid drug-related toxicities, drug monitoring and an individual dose adjustment should be considered because of the variability in busulfan bioavailability.