bluuebunny/crossref_metadata_2025 huggingface dataset:
Plasminogen activator inhibitor-1 secretion of endothelial cells increases fibrinolytic resistance of an in vitro fibrin clot: evidence for a key role of endothelial cells in thrombolytic resistance
Authors: Handt S , Jerome WG , Tietze L , Hantgan RR
Published: October 2019

Time-dependent thrombolytic resistance is a critical problem in thrombolytic therapy for acute myocardial infarction. Platelets have been regarded as the main source of plasminogen activator inhibitor-1 (PAI-1) found in occlusive platelet-rich clots. However, endothelial cells are also known to influence the fibrinolytic capacity of blood vessels, but their ability to actively mediate time-dependent thrombolytic resistance has not been fully established. We will show that, in vitro, tumor necrosis factor-alpha-stimulated endothelial cells secrete large amounts of PAI-1 over a period of hours, which then binds to fibrin and protects the clot from tissue plasminogen activator- induced fibrinolysis. In vivo, endothelial cells covering atherosclerotic plaques are influenced by cytokines synthesized by plaque cells. Therefore, we propose that continuous activation of endothelial cells in atherosclerotic blood vessels, followed by elevated PAI-1 secretion and storage of active PAI-1 in the fibrin matrix, leads to clot stabilization. This scenario makes endothelial cells a major factor in time-dependent thrombolytic resistance.
Correction of the platelet adhesion defect in delta-storage pool deficiency at elevated hematocrit--possible role of adenosine diphosphate
Authors: Weiss HJ , Lages B , Hoffmann T , Turitto VT
Published: October 2019

Previous studies on patients with storage pool deficiency (SPD) who are specifically deficient in platelet dense granules (delta-SPD) have suggested a role for dense granule substances, in all likelihood adenosine diphosphate (ADP), in mediating thrombus formation on subendothelium at high shear rates. The role of dense granule substances in mediating platelet adhesion appears to be more complicated Previous studies in delta-SPD suggested an adhesion defect that was strongly influenced by the patient's hematocrit (Hct) value. To explore further the possibility that red blood cells (RBCs) may influence the role that platelet storage granules play in mediating adhesion at high shear rates, we have measured adhesion (and thrombus formation) throughout a preselected range of Hct values (30% to 60%) in normal subjects and in patients with delta-SPD. The present studies confirm the defect in platelet adhesion in patients with delta-SPD, most significantly at Hct values of 30% to 40%. This defect (but not that of thrombus formation) can be completely corrected by the addition of RBCs. The correction of the platelet adhesion defect by RBCs was specific for delta-SPD; it was not observed in either von Willebrand's disease or thrombasthenia. Studies performed on normal blood under conditions that could be expected to block any effect of ADP on adhesion and an analysis of the type of adhesion defect in delta-SPD suggest that ADP may be involved in the process required for platelet spreading on the subendothelium. The corrective effect of RBCs on platelet adhesion in delta-SPD appears to be chemical rather than physical in nature, possibly due to shear-induced release of RBC ADP or to other recently described properties of RBCs that enhance collagen- induced platelet interactions.
Differential coupling of CC chemokine receptors to multiple heterotrimeric G proteins in human interleukin-2-activated natural killer cells
Authors: al-Aoukaty A , Schall TJ , Maghazachi AA
Published: October 2019

Using two different approaches, we have investigated the types of G proteins coupled to CC chemokine receptors. First, permeabilization of interleukin-2-activated natural killer (IANK) cells with streptolysin-O and introduction of anti-G protein antibodies inside these cells resulted in the following. (1) Anti-G(s), anti-G(o), and anti-G(z) inhibited the migration of IANK cells in response to macrophage- inflammatory protein-1 alpha (MIP-1 alpha), monocyte chemoattractant protein-1 (MCP-1), or regulated on activation normal T cell expressed and secreted (RANTES). (2) Anti-Gi inhibited their migration in response to MCP-1 or RANTES but not in response to MIP-1 alpha. Second, incubation of IANK cell membranes with anti-G protein antibodies before incubating with (gamma-35S) GTP or (gamma-32P) GTP, resulted in the following. (1) Anti-G(s), anti-G(o), or anti-G(z) inhibited GTP binding and GTPase activity in the presence of MIP-1 alpha, or RANTES. (2) Anti- G(i) inhibited GTP binding and GTPase activity in the presence of MCP-1 or RANTES but not in the presence of MIP-1 alpha. The inhibitory effect of anti-G protein antibodies was reversed upon incubating these antibodies with their respective synthetic peptides before addition to IANK cell membranes. These results suggest that MCP-1 and RANTES receptors are promiscuously coupled to multiple G proteins in IANK cell membranes and that this coupling is different from MIP-1 alpha receptors, which seem to be coupled to G(s), G(o), and G(z) but not to G(i).
High-level nuclear NF-kappa B and Oct-2 is a common feature of cultured Hodgkin/Reed-Sternberg cells
Authors: Bargou RC , Leng C , Krappmann D , Emmerich F , Mapara MY , Bommert K , Royer HD , Scheidereit C , Dorken B
Published: October 2019

There is a considerable lack of understanding about the common molecular defects that form the basis for the occurrence of Hodgkin's lymphoma. Despite a number of molecular tools used thus far in immunophenotypic and karyotypic studies, it has not been possible to establish a single common trait among various Hodgkin (H)-cell lines or primary tumor cells that would allow classification into a particular hematopoietic lineage. With this study, we demonstrate that a characteristic expression pattern of transcription factors provides a unifying principle. Seven different cell lines derived from patients with Hodgkin's disease (HD), as well as primary H/Reed-Sternberg (RS) cells isolated from the pericardial fluid of a patient with HD, were compared with a number of hematopoietic and nonhematopoietic cell lines for the expression of Oct-2, a tissue-specific transcription factor normally restricted to B cells, and nuclear factor kappa B (NF-kappa B), an inducible transcription factor. Regardless of the heterogeneous phenotypes and genotypes of the H cell lines, which varied inconsistently between B-cell-, T-cell-, or monocyte-like properties, all H cells tested displayed expression of Oct-2 protein at levels comparable to those seen in B cells. Furthermore, all cell lines showed an abundant constitutive nuclear NF-kappa B activity. Interestingly, anaplastic large-cell lymphoma (ALCL) cell lines, which have many features in common with H/RS cells, were devoid constitutive nuclear NF- kappa B activity. Unlike the constitutive NF-kappa B activity known for B cells, which mainly consists of the p50 and c-Rel or RelB subunits, we demonstrate by antibody supershifting experiments that H cells contain constitutive nuclear p50 and p65, the dimeric NF-kappa B normally observed only for limited time intervals after stimulation with diverse inducers. Additionally, some H-cell lines also displayed nuclear c-Rel activity, whereas RelB or p52 were not detected as part of the constitutive activity. The expression pattern of Oct-2 and NF- kappa B appears to be a unifying and characteristic property of H cells and might explain the deregulated expression of various cytokines leading to the clinical and pathologic manifestations of HD.
Bispecific antibody-mediated immunotherapy of the BCL1 lymphoma: increased efficacy with multiple injections and CD28-induced costimulation
Authors: Demanet C , Brissinck J , De Jonge J , Thielemans K
Published: October 2019

The BCL1 lymphoma in Balb/c mice can be successfully treated with bispecific (anti-CD3 x anti-idiotype) antibodies (BSABs). In these experiments, animals were injected intraperitoneally (IP) with 5 x 10(3) tumor cells (day 0) and treated with one single intravenous (IV) injection of 5 micrograms BSAB (day 9). Because cross-linking of the CD3 complex is not in itself sufficient to activate resting T cells, the therapeutic success was mainly based on the progressive retargeting of the relatively small cytotoxic T-lymphocyte effector cell pool already in existence in vivo. For this reason, the therapy lost its effectiveness at higher tumor loads. Two possibilities were explored to treat mice with a higher tumor load (10(5) tumor cells). First multiple injections of BSABs were used, but a dose-related monovalent anti-CD3 immunosuppression was induced. This approach was only beneficial when the immune system was able to recover from the previous injection of BSAB. As a second approach, CD28 costimulation together with BSABs were used in an attempt to activate resting T cells, ultimately enlarging the effector T-cell pool. However, we were repeatedly unsuccessful in attempts to improve our in vivo results using young, naive animals in which the majority of T cells are of the naive phenotype. Only when animals were primed to induce the memory T-cell type was a significantly better outcome observed, and then only with multiple injections of BSABs and anti-CD28 monoclonal antibodies (MoAbs), rather than with BSAB or anti-CD28 MoAb alone. These results suggest that this combination was able to activate memory and effector T cells and to focus them on the tumor, but was unable to activate naive T cells fully. The in vivo potency of the BSAB and CD28 costimulation was shown by the fact that one-tenth of the quantity of BSAB was required to cure animals with 20 times the tumor load.
Infrequent alterations of the RAR alpha gene in acute myelogenous leukemias, retinoic acid-resistant acute promyelocytic leukemias, myelodysplastic syndromes, and cell lines
Authors: Morosetti R , Grignani F , Liberatore C , Pelicci PG , Schiller GJ , Kizaki M , Bartram CR , Miller CW , Koeffler HP
Published: October 2019

Retinoids are important regulators of cell growth and differentiation in vitro and in vivo and they exert their biologic activities by binding to nuclear retinoic acid receptors (RARs; alpha, beta, and gamma) and retinoid X receptors (RXRs; alpha, beta, and gamma). All- trans retinoic acid (RA) induces complete remission in patients with acute promyelocytic leukemia (APL) presumably by binding directly to RAR alpha of APL cells. Leukemic blasts from APL patients initially responsive to RA can become resistant to the agent. HL-60 myeloblasts cultured with RA have developed mutations of the ligand-binding region of RAR alpha and have become resistant to RA. Furthermore, insertion of an RAR alpha with an alteration in the ligand-binding region into normal murine bone marrow cells can result in growth factor-dependent immortalization of the early hematopoietic cells. To determine if alterations of the ligand binding domain of RAR alpha might be involved in several malignant hematologic disorders, the mutational status of this region (exons 7, 8, and 9) was examined in 118 samples that included a variety of cell lines and fresh cells from patients with myelodysplastic syndromes (MDS) and acute myeloid leukemias (AML), including 20 APL patients, 5 of whom were resistant to RA and 1 who was refractory to RA at diagnosis, using polymerase chain reaction-single- strand conformational polymorphism (PCR-SSCP) analysis and DNA sequencing. In addition, 7 of the 20 APLs were studied for alterations of the other coding exons of the gene (exons 2 through 6). No mutations of RAR alpha were detected. Although the sensitivity of PCR-SSCP analysis is less than 100%, these findings suggest that alterations of RAR alpha gene are rare and therefore other mechanisms must be involved in the onset of resistance to retinoids and in the lack of differentiation in disorders of the myeloid lineage.
Protein phosphorylation in neutrophils from patients with p67-phox- deficient chronic granulomatous disease
Authors: Heyworth PG , Ding J , Erickson RW , Lu DJ , Curnutte JT , Badwey JA
Published: October 2019

Neutrophils are known to contain a major 67-kD protein that undergoes enhanced phosphorylation and translocation to the membrane during cell stimulation. Recent studies have assumed that this 67-kD phosphoprotein is the 67-kD subunit of the phagocyte oxidase (p67-phox). We compare here the protein phosphorylation patterns in lysates of normal neutrophils and neutrophils from patients with chronic granulomatous disease (CGD) that are completely deficient in p67-phox. The phosphoproteins were labeled by incubation of the cells with radioactive inorganic phosphate (32Pi) or by the addition of [gamma- 32P]ATP to electropermeabilized neutrophils. With either method, stimulation of the normal or CGD cells always resulted in an enhanced incorporation of 32p into two proteins in the 67-kD area. The extent of phosphorylation of these two proteins was very similar in the normal and CGD cells when permeabilized neutrophils loaded with [gamma - 32P]ATP were compared. Moreover, no overall differences in the protein phosphorylation patterns were observed between the normal and CGD cells. Our data indicate that the major 67-kD phosphoproteins observed in stimulated neutrophils are clearly different from p67-phox.
Sickle erythrocytes adhere to polymorphonuclear neutrophils and activate the neutrophil respiratory burst
Authors: Hofstra TC , Kalra VK , Meiselman HJ , Coates TD
Published: October 2019

The vasoocclusive process in patients with sickle cell disease (SCD) is complex and involves interactions among sickle erythrocytes (SS-RBC), vascular endothelium, and plasma and cellular components. The role of neutrophils (PMN) in vasoocclusion has not been examined. Patients with SCD appear to have chronically activated PMN. Because the first step in PMN activation is particle recognition, we explored whether normal PMN recognize SS-RBC and whether this recognition results in PMN monolayers, significantly more SS-RBC adhered to the PMN than did normal erythrocytes (AA-RBC; P < .001). Preincubation of erythrocytes with autologous plasma significantly increased the adherence of SS-RBC to PMN but had no effect on AA-RBC (P < .001). When adhesion of density fractionated SS-RBC was performed, dense SS-RBC showed greater adherence to the PMN monolayers than did light SS-RBC (P < .001). To determine mechanisms of this adhesion, IgG and Arg-Gly-Asp-Ser (RGDS) receptor sites on PMN were saturated. IgG inhibited adherence of dense SS-RBC, whereas RGDS inhibited adherence in both fractions, although to a greater extent in the light fraction. We measured SS-RBC activation of PMN by incubating SS-RBC with 2′, 7′-Dichloro-fluroescin Diacetate (DCF)-labeled PMN. Incubation of PMN with SS-RBC resulted in a significant increase in fluorescence compared to AA-RBC. We show here that PMN recognize SS-RBC through multiple mechanisms and that this recognition results in activation of PMN. These findings contribute to the understanding of vasoocclusive crisis in patients with SCD and may have therapeutic implications.
The differentiation and maturation mediator for human myeloid leukemia cells shares homology with neuroleukin or phosphoglucose isomerase
Authors: Xu W , Seiter K , Feldman E , Ahmed T , Chiao JW
Published: October 2019

The identity of the maturation inducer capable of mediating the differentiation of human myeloid leukemic HL-60 calls to terminal monocytic cells was investigated. One of such inducers from T cells was purified as a 54.3-kD peptide. The amino acid sequence of a tryptic peptide and the enzyme cleavage sites revealed 100% homology to neuroleukin or phosphoglucose isomerase (PGI). Neuroleukin mediates differentiation of neurons and is homologous to PGI, which catalyzes the interconversion of glucose-6-phosphate and fructose-6-phosphate. The 54.3-kD inducer was shown to have PGI enzymatic activity. Separately purified PGI by substrate-elution exhibited identical specificity as the maturation inducer for HL-60 cell differentiation. They mediated a reduction of proliferating S and G2M cells, and the mature monocytic calls acquired complement receptors, phagocytic capacity, and adherence morphology. The magnitude of differentiation was dosage dependent on the inducer, with a bell-shaped curve. At the excess dose range cells did not undergo differentiation and remained in a proliferating cycle. Abnormally elevated PGI enzyme activities were detected in the plasma of acute myelogenous leukemia patients. Whether they represent an excess of the differentiation regulator in patients and are important in leukemogenesis remain to be investigated.
Dendritic cells and macrophages can mature independently from a human bone marrow-derived, post-colony-forming unit intermediate
Authors: Szabolcs P , Avigan D , Gezelter S , Ciocon DH , Moore MA , Steinman RM , Young JW
Published: October 2019

CD34+ precursors in normal human bone marrow (BM) generate large numbers of dendritic cells alongside macrophages and granulocytic precursors when cultured for 12 to 14 days in c-kit ligand, granulocyte- macrophage colony-stimulating factor (GM-CSF), and tumor necrosis factor-alpha (TNF-alpha). This study reports an intermediate cell type that develops by day 6, and has the potential to differentiate into either macrophages or dendritic cells. When the d6 progeny are depleted of mature macrophages and residual CD34+ precursors, a discrete CD14+ HLA-DR+ population persists in addition to immunostimulatory CD14- HLA- DR() dendritic cells. Half of the CD14+ HLA-DR+ population is in cell cycle (Ki-67+), but colony-forming units (CFUs) are no longer detectable. The calls are c-fms+, but lack myeloperoxidase and nonspecific esterase. They also possess substantial phagocytic and allostimulatory activity. These post-CFU, CD14+ HLA-DR+ intermediates develop into typical macrophages when recultured in the absence of exogenous cytokines. M-CSF supports up to approximately 2.5-fold expansion of macrophage progeny. In contrast, the combination of GM-CSF and TNF-alpha supports quantitative differentiation into dendritic cells, lacking c-fms, CD14, and other macrophage properties, and expressing HLA-DR, CD1a, CD83, CD80, CD86, and potent allostimulatory activity. Therefore, normal CD34+ BM precursors can generate a post-CFU bipotential intermediate in the presence of c-kit ligand, GM-CSF, and TNF-alpha. This intermediate cell type will develop along the dendritic cell pathway when macrophages are removed and GM-CSF and TNF-alpha are provided. Alternatively, it can differentiate along a macrophage pathway when recultured with or without M-CSF.
Structural and functional differentiation of sinusoidal endothelial cells during liver organogenesis in humans
Authors: Couvelard A , Scoazec JY , Dauge MC , Bringuier AF , Potet F , Feldmann G
Published: October 2019

During fetal life, human liver sinusoids, which differentiate between 4 and 12 weeks of gestation from capillaries of the septum transversum, must support an important hematopoietic function and acquire the structural and functional characteristics of adult sinusoids. To gain insight into their differentiation process, we studied the expression of (1) markers of continuous endothelia, absent from adult sinusoidal endothelial cells (PECAM-1, CD34, and 1F10); (2) functional markers of adult sinusoidal endothelial calls (CD4, 1CAM-1, CD32, and CD14); and (3) extracellular matrix components (laminin, tenascin, fibronectin, and thrombospondin) in 37 fetuses of different gestational ages. We identified two successive differentiation events. (1) An early structural differentiation, occurring from 5 to 12 weeks of gestation, was characterized by the loss of continuous endothelial cell markers and a reduction in the perisinusoidal amount of laminin and in the deposition of tenascin, fibronectin, and thrombospondin; at the end of this process, fetal liver sinusoids present structural characteristics comparable to those of the sinuses in adult hematopoietic bone marrow. (2) A later functional differentiation was characterized by the acquisition of the markers of adult sinusoidal endothelial cells, initiating at 10 weeks of gestation and completed by 20 weeks of gestation; this process likely contributes to adapt liver sinusoids to the specific functions of the adult hepatic tissue.
Sustained expression of therapeutic levels of human factor VIII in mice
Authors: Connelly S , Gardner JM , Lyons RM , McClelland A , Kaleko M
Published: October 2019

Deficiency of coagulation factor VIII (FVIII) results in hemophilia A, a common hereditary bleeding disorder. Using a human FVIII-encoding adenoviral vector, Av1ALAPH81, we have demonstrated expression of therapeutic levels of human FVIII in mice sustained for more than 5 months after vector administration. Administration of a high dose (4 x 10(9) plaque-forming units [pfu]) of Av1ALAPH81 to mice resulted in a peak expression of 2,063 ng/mL of human FVIII in the mouse plasma, with levels decreasing to background by weeks 15 to 17. Normal FVIII levels in humans range from 100 to 200 ng/mL and therapeutic levels are as low as 10 ng/mL. Alternatively, administration of 8- to 80-fold lower vector doses (5 x 10(8) pfu to 5 x 10(7) pfu) to normal adult mice resulted in expression of FVIII at therapeutic levels sustained for at least 22 weeks. Detailed analysis of vector toxicity indicated that the high vector dose caused a dramatic elevation of liver-specific enzyme levels, whereas an eight-fold lower vector dose was significantly less hepatotoxic. The data presented here demonstrate that administration of lower, less toxic vector doses allow long-term persistence of FVIII expression.
Quality control of multidrug resistance assays in adult acute leukemia: correlation between assays for P-glycoprotein expression and activity
Authors: Broxterman HJ , Sonneveld P , Feller N , Ossenkoppele GJ , Wahrer DC , Eekman CA , Schoester M , Lankelma J , Pinedo HM , Lowenberg B , Schuurhuis GJ
Published: October 2019

We have compared multiple assays for the P-glycoprotein (Pgp/MDR1) phenotype in fresh and thawed adult acute leukemia to validate and quantitate measures for the expression and function of Pgp. The results are related to the Pgp-expressing KB8 and KB8–5 call lines. The most sensitive assay was the measurement of modulation of the rhodamine 123 (R123) fluorescence by 2 micromol/L PSC833, followed by the modulation of the probe calcein-AM. We also found a good intralaboratory and interlaboratory correlation between the values of the R123/PSC833 assay for fresh as well as thawed samples. In addition, the affects of PSC833 on 3H-daunorubicin (DNR) accumulation, DNR fluorescence, and 3H- vincristine accumulation were very similar. The correlation between the DNR/PSC833 and R123/PSC833 test was r = .86 (N = 51). The modulation of drug accumulation by 8 micromol/L verapamil was the some as the PSC833 effect for DNR (117%, N = 21), but was higher for vincristine in every single case (161% v 121%, N = 22; P< .001), indicating additional verapamil effects, not related to Pgp. The correlation of the staining of viable cells for Pgp with the monoclonal antibody MRK16 was r = .77 (N = 52) for the R123/PSC833 functional test and r = .84 (N = 50) for the DNR/PSC833 test. From these results it could be calculated that a maximal increase of the mean DNR accumulation of about 50% can be achieved by blocking Pgp pump activity with PSC833 in leukemic blast samples with the highest mean Pgp expression. Subpopulations of blast calls with higher Pgp activity are likely to be present. Their relevance has to be studied further. The methods outlined here allow the reliable, quantitative monitoring of the Pgp/MDR1 phenotype in leukemias in multicentered, clinical Pgp modulation studies.
Defective iron supply for erythropoiesis and adequate endogenous erythropoietin production in the anemia associated with systemic-onset juvenile chronic arthritis
Authors: Cazzola M , Ponchio L , de Benedetti F , Ravelli A , Rosti V , Beguin Y , Invernizzi R , Barosi G , Martini A
Published: October 2019

Systemic-onset juvenile chronic arthritis (SoJCA) is associated with high levels of circulating interleukin-6 (IL-6) and is frequently complicated by severe microcytic anemia whose pathogenesis is unclear. Therefore, we studied 20 consecutive SoJCA patients with hemoglobin (Hb) levels <12 g/dL, evaluating erythroid progenitor proliferation, endogenous erythropoietin production, body iron status, and iron supply for erythropoiesis. Hb concentrations ranged from 6.5 to 11.9 g/dL. Hb level was directly related to mean corpuscular volume (r = .82, P < .001) and inversely related to circulating transferrin receptor (r = - .81, P < .001) suggesting that the severity of anemia was directly proportional to the degree of iron-deficient erythropoiesis. Serum ferritin ranged from 18 to 1,660 microgram/L and was unrelated to Hb level. Bone marrow iron stores wore markedly reduced in the three children investigated, and they also showed increased serum transferrin receptor and normal-to-high serum ferritin. All 20 patients had elevated IL-6 levels and normal in vitro growth of erythroid progenitors. Endogenous erythropoietin (epo) production was appropriate for the degree of anemia as judged by both the observed to predicted log (serum epo) ratio 10.95 +/- 0.12) and a comparison of the serum epo- Hb regression found in these subjects with that of thalassemia patients. Multiple regression analysis showed that serum transferrin receptor was the parameter most closely related to hemoglobin concentration: variation in circulating transferrin receptor explained 61% of the variation in Hb level (P < .001). In 10 severely anemic patients, amelioration of anemia following intravenous iron administration resulted in normalization of serum transferrin receptor. Defective iron supply to the erythron rather than blunted epo production is the major cause of the microcytic anemia associated with SoJCA. A true body-iron deficiency caused by decreased iron absorption likely complicates long-lasting inflammation in the most anemic children, and this can be recognized by high serum transferrin receptor levels. Although oral iron is of no benefit, intravenous iron saccharate is a safe and effective means for improving iron availability for erythropoiesis and correcting this anemia. Thus, while chronically high endogenous IL-6 levels do not appear to blunt epo production, they are probably responsible for the observed abnormalities in iron metabolism. Anemia of chronic disease encompasses a variety of anemic conditions whose peculiar features may specifically correlate with the type of cytokine(s) predominantly released.
Glycoprotein IV-independent adhesion of sickle red blood cells to immobilized thrombospondin under flow conditions
Authors: Joneckis CC , Shock DD , Cunningham ML , Orringer EP , Parise LV
Published: October 2019

The abnormal adherence of red blood cells (RBC to the blood vessel wall is believed to contribute to the vascular occlusion observed in patients with sickle call anemia. The cell adhesion receptors GPIV (CD36) and integrin alpha 4 beta 1 (CD49d/CD29) were previously identified on circulating sickle reticulocytes, and shown to mediate sickle RBC adhesion to the endothelium. The presence of damaged endothelium in these patients suggests that exposed extracellular matrix proteins could provide a potential substrate for sickle RBC adhesion. To determine whether RBC adhesion receptors could mediate adhesion to extracellular matrix proteins, we tested their ability to adhere to a variety of immobilized, purified proteins under flow conditions. Neither sickle nor normal RBC adhered to fibronectin, vitronectin, fibrinogen, or collagen. In contrast, we observed substantial adhesion of sickle but not normal RBC to thrombospondin (TSP). The adhesion was not inhibited with known antagonists of the GPIV-TSP interaction, nor by inhibitors of several other known binding domains in TSP. Moreover, the adhesion was resistant to inhibition by soluble TSP, suggesting that immobilization of TSP exposes an adhesive site that is cryptic on TSP in solution. However, the glycosaminoglycans, chondroitin sulfate A, and dextran sulfate were potent inhibitors of this adhesion. These results suggest that a mechanism distinct from GPIV is responsible for sickle RBC adhesion to immobilized TSP under flow conditions.
Transplantation of allogeneic CD34+ blood cells
Authors: Link H , Arseniev L , Bahre O , Kadar JG , Diedrich H , Poliwoda H
Published: October 2019

Pluripotent stem cells of hematopoiesis and lymphopoiesis are among the CD34+ cells in blood or bone marrow. After granulocyte-colony stimulating factor (G-CSF) treatment, 1% to 2% of the mononuclear cells in blood are CD34+ cells, which can be procured by leukapheresis. We investigated the potential of CD34+ blood cells for reconstituting hematopoiesis and lymphopoiesis after allogeneic transplantation. HLA- identical sibling donors of 10 patients with hematologic malignancies were treated with G-CSF (filgrastim), 5 microgram/kg subcutaneously twice daily for 5 to 7 days. CD34+ cells were selected from the apheresis concentrates by immunoadsorption, concomitantly the number of T cells was reduced 100- to 1,000-fold. After transplantation, five patients received cyclosporine A for graft-versus-host disease (GvHD) prophylaxis (group I); five patients additionally received methotrexate (group II). G-CSF and erythropoietin were given to all patients. Mean numbers of 7.45 x 10(6) CD34+ and 1.2 x 10(6) CD3+ cells per kilogram were transplanted. In group I, the median times of neutrophil recovery to 100, 500, and 1,000 per mm3 were 10, 10, and 11 days, respectively. Group II patients reached these neutrophil levels after 10, 14, and 15 days, respectively. Platelet transfusions were administered for a median of 18 days in group I and 30 days in group II, and red blood cells for 9 and 12 days, respectively. Between day 30 and 60, lymphocytes reached levels of 353 +/- 269 cells per mm3. The median grades of acute GvHD were III in group I and I in group II. Two patients in group I died from acute GvHD. Two leukemic relapses occurred in group II. Complete and stable donor hematopoiesis was shown in all patients with a median follow up of 370 (45 to 481) days. Allogeneic blood CD34+ cells can successfully reconstitute hematopoiesis and lymphopoiesis. Reduction of T cells by CD34+ blood cell enrichment and cyclosporine A alone might not be sufficient for prophylaxis of severe acute GvHD.
Preferential hematopoiesis by paroxysmal nocturnal hemoglobinuria clone engrafted in SCID mice
Authors: Iwamoto N , Kawaguchi T , Horikawa K , Nagakura S , Kagimoto T , Suda T , Takatsuki K , Nakakuma H
Published: October 2019

In paroxysmal nocturnal hemoglobinuria (PNH), little is known about the molecular events leading to the clinical manifestations except for the hemolysis. To unfold the complex pathophysiology, it is necessary to elucidate the nature of the PNH clone. PNH exhibits an acquired stem cell disorder, a clonal expansion of affected cells, concomitant depression of normal hematopoiesis in bone marrow (BM), and, although infrequently, the development of leukemia. The PNH clone is thus expected to exhibit some neoplastic features. We report here that CD34+ hematopoietic progenitor cells of PNH-BM yielded blood cells of three lineages with PNH phenotype alone when transplanted into sublethally irradiated severe combined immunedeficient mice. The hematopoiesis persisted for more than 10 months and did not always need human cytokines. In contrast, the hematopoiesis by control grafts obtained from healthy volunteers required an intense cytokine treatment. This in vivo model defines the preferential hematopoiesis of pluripotent PNH progenitor cells, indicating the intrinsic growth abnormality of PNH clone.
Involvement of nitric oxide in target-cell lysis and DNA fragmentation induced by murine natural killer cells
Authors: Filep JG , Baron C , Lachance S , Perreault C , Chan JS
Published: October 2019

Although it has been recognized for sometime that target cells destroyed by natural killer (NK) cells die largely by apoptosis, the underlying mechanisms are not fully understood. The aim of the present study was to examine the role of nitric oxide (NO) in mediating murine NK-cell-induced killing of YAC-1 lymphoma cells. NK calls induced extensive release of 125I-DNA and 51Cr from YAC-1 cells. The target killing ability of NK cells was associated with an increased production of NO as measured by concentrations of nitrite in the culture medium. That YAC-1 killing resulted, in part, from the production of NO was confirmed by the significant protection of cell lysis in L-arginine- depleted medium and by approximately 30 % attenuation of cell lysis and DNA fragmentation by an inhibitor of NO synthase, NG-nitro-L-arginine methyl ester (L-NAME) in a culture medium containing 1 mmol/L L- arginine. Fluorescence microscopic examination of YAC-1 cells showed the presence of changes in nuclear morphology characteristic for apoptosis. The percentage of apoptotic cells was markedly decreased by L-NAME. Further evidence for apoptosis is provided by the specific pattern of internucleosomal DNA fragmentation both in the absence and presence of L-NAME. During target-cell killing, an increased oxidation of intracellularly trapped dichlorofluorescein was observed in cells labeled with an antimouse NK-cell monoclonal antibody, as measured by flow cytometry. These increases were effectively prevented by L-NAME, but not W-13, an inhibitor of calmodulin. The ability of NO to induce cell lysis and DNA fragmentation in YAC-1 cells was further demonstrated by exposing tumor cells to chemically generated NO. Taken together, these observations suggest a role for NO as one of the mediators of NK-cell-mediated DNA fragmentation and cell lysis.
Id2 expression increases with differentiation of human myeloid cells
Authors: Ishiguro A , Spirin KS , Shiohara M , Tobler A , Gombart AF , Israel MA , Norton JD , Koeffler HP
Published: October 2019

Id proteins are helix-loop-helix (HLH) transcriptional factors that lack the basic DNA binding domain. The Id proteins have been reported generally to function as inhibitors of cell differentiation, and their gene expression is often downregulated during cell differentiation. We examined the expression of human Id mRNAs by Northern hybridization in 11 human myeloid cell lines, several myeloid cell lines induced to differentiate, fresh myeloid leukemia samples, and normal human myeloid cells. Id2 mRNA was expressed in myelomonoblastic and monoblastic leukemic cells (PLB-985, THP-1, and U-937) but was weakly expressed in myeloblastic leukemic cells (KG-1 and HL-60). Id2 mRNA levels markedly increased with induction of differentiation of myeloid blasts (HL-60, PLB-985, THP-1, and U-937) toward either granulocytes or macrophages. Examination of fresh acute myeloid leukemic samples from 22 individuals also showed prominent Id2 mRNA expression in those samples having more differentiated blasts. Using the French-American-British classification, only 2 of 8 M0/M1 samples expressed Id2 mRNA; however, 10 of 13 M2/M3/M4 samples expressed it. In normal human myeloid cells, Id2 mRNA was expressed in cultured macrophages from bone marrow and in mature granulocytes and monocytes from peripheral blood. The half-life of Id2 mRNA was short (1 hour), and its expression was inducible by cessation of protein synthesis. Id3 mRNA was moderately expressed in monoblastic cell lines (THP-1 and U-937), and levels decreased with their differentiation. Almost no Id3 expression was detectable in either other myeloid leukemia lines, fresh leukemic samples, or normal human myeloid cells by Northern analyses. Id1 mRNA was not detected by polymerase chain reaction in either leukemic or normal myeloid cells except in K562 myeloid/erythroid cells. These results showed that Id2 mRNA was constitutively expressed in more mature myeloid blast cells and level markedly increased with terminal myeloid differentiation, suggesting that Id2 protein may inhibit an HLH transcriptional complex that normally represses myeloid differentiation.
Relationship between minimal residual disease and outcome in adult acute lymphoblastic leukemia
Authors: Brisco J , Hughes E , Neoh SH , Sykes PJ , Bradstock K , Enno A , Szer J , McCaul K , Morley AA
Published: October 2019

In children with acute lymphoblastic leukemia (ALL), the level of minimal residual disease (MRD) at the end of induction strongly predicts outcome, presumably because it measures both drug sensitivity and the number of leukemic cells requiring elimination. Children with high levels (> 10(-3) leukemic cells per marrow cell) nearly always relapse, whereas those with low levels (<2 x 10(-5)) seldom do. However, the importance of MRD in adult ALL is unclear. We studied 27 patients aged 14 to 74 who were treated with a standard protocol and who attained morphological remission. MRD in the marrow at first remission was quantified by using the polymerase chain reaction (PCR), with the rearranged immunoglobulin heavy chain gene as a molecular marker. Levels of MRD varied from 3 x 10(-1) to <7 x 10(-7). The probability of long-term relapse-free survival was significantly related to the level of MRD and only one of nine patients with MRD >10(- 3) did not relapse. For patients who did relapse, there was an inverse relationship between MRD level and the length of remission. Overall, MRD in adults in whom a translocation had not been identified was significantly higher than in comparably-treated children, suggesting that ALL in adults is more drug-resistant than in children.
Development of day-8 colony-forming unit-spleen hematopoietic progenitors during early murine embryogenesis: spatial and temporal mapping
Authors: Medvinsky AL , Gan OI , Semenova ML , Samoylina NL
Published: October 2019

The ontogeny of the hematopoietic system in mammalian embryos occurs during the yolk sac (YS) and the fetal liver (FL) stages. Events leading to the establishment of hematopoiesis in the FL remain obscure. The appearance of colony-forming units-spleen (CFU-S) in the FL is preceded by a gradual increase of CFU-S in the YS and a more rapid increase in the AGM region (area comprising dorsal aorta, gonads, and mesonephros) during day 10 of development (Medvinsky et al, Nature 364:64, 1993). By this time, the AGM CFU-S attain a high frequency equivalent to that found in the adult bone marrow. The analogous area gives rise to adult hematopoiesis in amphibians and probably in birds. We present here a more complete picture of CFU-S development during transition from the pre-liver to liver stage of hematopoiesis. (1) Dissectional analysis of the mouse AGM region shows the presence of CFU- S both around the dorsal aorta and in the uro-genital ridges. (2) The embryonic gut also shows low but distinctive CFU-S activity. This initial intrabody pattern of CFU-S distribution in murine embryogenesis parallels that found for primordial germ cells. (3) The beginning of definitive liver hematopoiesis is accompanied by wide dissemination of CFU-S in the embryonic tissues. (4) Comparison of spleen colonies arising from the AGM and YS has shown morphologic differences. In contrast to simple erythroid constitution of the YS colonies, a broader variety of cells are found within the AGM-derived colonies that are similar to those derived from 11-day FL. These data suggest a lineage relationship for hematopoietic progenitors between the AGM region and the FL.
Acceleration of hematopoietic reconstitution with a synthetic cytokine (SC-55494) after radiation-induced bone marrow aplasia
Authors: Farese AM , Herodin F , McKearn JP , Baum C , Burton E , MacVittie TJ
Published: October 2019

The synthetic cytokine (Synthokine) SC-55494 is a high-affinity interleukin-3 (IL-3) receptor ligand that stimulates greater in vitro multilineage hematopoietic activity than native IL-3, while inducing no significant increase in inflammatory activity relative to native IL-3. The aim of this study was to investigate the in vivo hematopoietic response of rhesus monkeys receiving Synthokine after radiation-induced marrow aplasia. Administration schedule and dose of Synthokine were evaluated. All animals were total-body irradiated (TBI) with 700 cGy 60Co gamma radiation on day 0. Beginning on day 1, cohorts of animals (n = 5) received Synthokine subcutaneously (SC) twice daily with 25 micrograms/kg/d or 100 micrograms/kg/d for 23 days or 100 micrograms/kg/d for 14 days. Control animals (n = 9) received human serum albumin SC once daily at 15 micrograms/kg/d for 23 days. Complete blood counts were monitored for 60 days postirradiation and the durations of neutropenia (NEUT; absolute neutrophil count [ANC] 500/microL) and thrombocytopenia (THROM; platelet count 20,000/microL) were assessed. Synthokine significantly (P .05) reduced the duration of THROM versus the HSA-treated animals regardless of dose or protocol length. The most striking reduction was obtained in the animals receiving 100 micrograms/kg/d for 23 days (THROM = 3.5 v 12.5 days in HSA control animals). Although the duration of NEUT was not significantly altered, the depth of the nadir was significantly lessened in all animal cohorts treated with Synthokine regardless of dose versus schedule length. Bone marrow progenitor cell cultures indicated a beneficial effect of Synthokine on the recovery of granulocyte-macrophage colony-forming units that was significantly higher at day 24 post-TBI in both cohorts treated at 25 and 100 micrograms/kg/d for 23 days relative to the control animals. Plasma pharmacokinetic parameters were evaluated in both normal and irradiated animals. Pharmacokinetic analysis performed in irradiated animals after 1 week of treatment suggests an effect of repetitive Synthokine schedule and/or TBI on distribution and/or elimination of Synthokine. These data show that the Synthokine, SC55 94, administered therapeutically post-TBI, significantly enhanced platelet recovery and modulated neutrophil nadir and may be clinically useful in the treatment of the myeloablated host.
LAF-4 encodes a lymphoid nuclear protein with transactivation potential that is homologous to AF-4, the gene fused to MLL in t(4;11) leukemias
Authors: Ma C , Staudt LM
Published: October 2019

A novel human gene, LAF-4, was isolated from a subtracted cDNA library that showed strong sequence similarity to AF-4, a gene that is translocated in t(4;11)(q21;q23) acute lymphoblastic leukemias (ALLs). In t(4;11) ALL, the AF-4 gene at 4q21 is translocated into the MLL locus at 11q23, resulting in the expression of an MLL/AF-4 fusion protein that is the presumptive oncoprotein. AF-4 and LAF-4 are homologous throughout their coding regions, yet neither protein is related to previously cloned genes. Human LAF-4 readily hybridized with genes in mouse and chicken, thus showing that this gene family has been highly conserved during vertebrate evolution. In mouse tissues, LAF-4 mRNA was found to be present at highest levels in lymphoid tissues, present at lower levels in brain and lung, and absent from other tissues. In human and mouse lymphoid cell lines, LAF-4 expression was highest in pre-B cells, intermediate in mature B cells, and absent in plasma cells, thus pointing to a potential regulatory role for LAF-4 in lymphoid development. Antibodies to LAF-4 showed it to be a nuclear protein that showed an uneven, granular immunofluorescence pattern. In vitro-translated LAF-4 was able to bind strongly to double-stranded DNA cellulose. Furthermore, both LAF-4 and AF-4 had domains that activated transcription strongly when fused to the GAL4 DNA-binding domain. Interestingly, the AF-4 transactivation domain is retained in the MLL/AF-4 fusion protein; thus, it may contribute to the transforming potential of the oncoprotein. Therefore, the cloning of LAF-4 has defined a new family of potential regulatory proteins that may function in lymphoid development and oncogenesis.
Moderate reduction of beta-globin gene transcript by a novel mutation in the 5' untranslated region: a study of its interaction with other genotypes in two families
Authors: Ho PJ , Rochette J , Fisher CA , Wonke B , Jarvis MK , Yardumian A , Thein SL
Published: October 2019

We have identified two individuals of Greek Cypriot origin with thalassemia intermedia. Molecular analysis has shown that each individual is a compound heterozygote for a previously described beta zero thalassemia allele and a novel mutation, C-->G in position +33, in the 5′ untranslated region of the beta globin gene. In both families the beta +33 allele is associated with the same beta haplotype (-++- ) suggesting that it is likely to be of a single origin, beta-cDNAs from normal and mutant beta alleles were isolated from peripheral blood reticulocytes using the technique of reverse transcription-polymerase chain reaction. Because the beta +33 (C-->G) mutation creates a cutting site for the restriction enzyme NlalV, we could demonstrate by differential restriction analysis that the beta gene with +33 mutation showed 25% to 35% residual activity compared with normal. The additive effect of this moderate deficit in beta globin production with the beta zero thalassemia mutation would explain the clinical phenotypes observed in the two probands. In contrast, two siblings of one proband who were also compound heterozygotes for the same beta thalassemia mutations, as well as heterozygotes for a nondeletional alpha thalassemia variant, and two other compound heterozygotes for the beta +33 and a beta+ thalassemia allele were completely asymptomatic. Individuals heterozygous for the beta +33 C-G mutation alone are clinically and hematologically silent, with normal red blood cell indices and normal levels of hemoglobin (Hb) A2. A direct relationship between genotypic and phenotypic severity is clearly demonstrated in these cases with obvious implications for prenatal diagnosis.
The t(5;17) variant of acute promyelocytic leukemia expresses a nucleophosmin-retinoic acid receptor fusion
Authors: Redner RL , Rush EA , Faas S , Rudert WA , Corey SJ
Published: October 2019

We have studied an acute promyelocytic leukemia (APL) patient with a variant t(5;17)(q32;q12). This translocation fuses the gene for the nucleolar phosphoprotein nucleophosmin (NPM) to the retinoic acid receptor alpha (RARA). Two alternatively spliced transcripts are expressed, which differ in 129 bases immediately upstream of the RARA sequence. The NPM sequences contained in the shorter NPM-RAR cDNA are identical to the NPM sequences contained in the NPM-ALK fusion gene expressed in t(2;5) lymphomas. The RARA sequences are the same as the RARA sequences found in the PML-RAR and PLZF-RAR fusion seen in t(15;17) and t(11;17) APL, respectively. Both NPM-RAR transcripts fuse NPM and RARA sequence in the same reading frame, to generate translation products of 57 kD and 62 kD. Both NPM-RAR proteins are expressed in the patient's leukemic cells, along with wild-type RARA derived from the uninvolved allele. In transcriptional assays using a retinoic acid response element reporter construct, both NPM-RAR fusion proteins act as retinoic acid-dependent transcriptional activators. This case defines a third class of APL rearrangements, all of which generate fusion proteins of RARA.