bluuebunny/crossref_metadata_2025 huggingface dataset:
Point mutations in the conserved box 1 region inactivate the human granulocyte colony-stimulating factor receptor for growth signal transduction and tyrosine phosphorylation of p75c-rel
Authors: Avalos BR , Hunter MG , Parker JM , Ceselski SK , Druker BJ , Corey SJ , Mehta VB
Published: October 2019

The human granulocyte colony-stimulating factor receptor (hG-CSFR) belongs to the cytokine receptor superfamily. As with other members of this family, the cytoplasmic domain of hG-CSFR lacks intrinsic tyrosine kinase activity. To identify critical regions mediating growth signal transduction by hG-CSFR, deletions or site-directed amino acid substitutions were introduced into the cytoplasmic domain of hG-CSFR, and the mutant cDNAs were transfected into the murine interleukin-3 (IL-3)-dependent Ba/F3 and FDCP cell lines. Truncation of the carboxy-terminal end of the receptor to the membrane-proximal 53 amino acids of the cytoplasmic domain, which retained the conserved Box 1 and Box 2 sequence motifs, decreased the ability of hG-CSFR to transduce G-CSF-mediated growth signals without an associated loss in receptor binding affinity. Substitution of proline by alanine at amino acid positions 639 and 641 within Box 1 completely abolished the G-CSF-mediated growth signal. Rapid induction of tyrosine phosphorylation of several cellular proteins, including a 75-kD protein (p75) identified as c-rel, was an early event associated with transduction of proliferative signals by hG-CSFR in Ba/F3 transfectants. Mutant receptors containing Pro-to-Ala substitutions that inactivated the receptor for mitogenic activity also inactivated the receptor for tyrosine-specific phosphorylation of p75. These results show that the conserved Box 1 sequence motif (amino acids 634 to 641) is critical for mitogenesis and activation of cellular tyrosine kinases by hG-CSFR.
Fas antigen expression on CD34+ human marrow cells is induced by interferon gamma and tumor necrosis factor alpha and potentiates cytokine-mediated hematopoietic suppression in vitro
Authors: Maciejewski J , Selleri C , Anderson S , Young NS
Published: October 2019

Activation of Fas antigen, a cell surface receptor molecule, by its ligand results in transduction of a signal for cell death. The Fas system has been implicated in target cell recognition, clonal development of immune effector cells, and termination of the cellular immune response. Fas antigen expression on lymphocytes is regulated by interferon gamma (IFN gamma) and tumor necrosis factor alpha (TNF alpha), cytokines that also have inhibitory effects on hematopoiesis. We investigated Fas antigen expression on human marrow cells and the effects of Fas activation on hematopoiesis in vitro. Freshly isolated immature hematopoietic cells, as defined by the CD34 marker, did not express Fas antigen at levels detectable by fluorescent staining. CD34+ cells, which include progenitors and stem cells, showed low levels of Fas expression in culture, even in the presence of growth factors. Stimulation by TNF alpha and IFN gamma markedly increased Fas antigen expression on CD34+ cells. Anti-Fas antibody, which mimics the action of the putative ligand, enhanced IFN gamma- and TNF alpha-mediated suppression of colony formation by bone marrow (BM) in a dose-dependent manner. This effect did not require the presence of accessory cells. Colony formation from mature (CD34+ CD38+) and immature (CD34+ CD38-) progenitor cells and long-term culture initiating cells were susceptible to the inhibitory action of anti-Fas antibody in the presence of IFN gamma and TNF alpha. Apoptosis assays performed on total BM cells and CD34+ cells showed that anti-Fas antibody induced programmed cell death of CD34+ BM cells.(ABSTRACT TRUNCATED AT 250 WORDS)
A peripheral blood-derived monolayer supports long-term cultures of human CD4+ and CD8+ T lymphocytes
Authors: Sutkowski N , Kuo ML , Amenta PS , Dougherty JP , Ron Y
Published: October 2019

An in vitro culture system has been developed for the long-term maintenance of primary, human peripheral blood and umbilical cord blood T lymphocytes, which does not rely on the use of stimulatory cytokines, antigen, or mitogens. In these cultures, a monolayer of adherent cells, some spindle-shaped and some resembling macrophages, developed within a week. All adherent cells were positive for the extracellular matrix proteins laminin and fibronectin, the intermediate filament vimentin, and for the surface markers major histocompatibility complex class II, platelet-endothelial cell adhesion molecule l (CD31), and E-Selectin (ELAM-1; CD62E). They were negative for the leukocyte common antigen (CD45), the macrophage marker MO-2 (CD14), muscle-specific actin, and Factor VIII-related antigen. These monolayers supported the maintenance of nonadherent, resting, mature T cells for up to 3 months, and these cells retained their ability to respond to mitogens and allogeneic cells. Both CD4+ and CD8+ cells were supported. The proportion of CD4+ and CD8+ cells remained unchanged after 3 months in culture. We have also used T cells from 2-month-old cultures as target cells for retroviral vector-mediated gene transfer. Up to 30% of the long-term T cells expressed the transferred lacZ gene after infection with a retroviral vector. The infection efficiency was similar to that obtained for fresh peripheral blood T cells, indicating that the long-term-cultured cells might be suitable for certain gene therapy applications.
Use of a water-soluble busulfan formulation--pharmacokinetic studies in a canine model
Authors: Ehninger G , Schuler U , Renner U , Ehrsam M , Zeller KP , Blanz J , Storb R , Deeg HJ
Published: October 2019

In a canine model we investigated the toxicity and pharmacokinetics of a water soluble busulfan preparation. Busulfan was dissolved in dimethylsulfoxide (DMSO) and administered either orally or intravenously in a single dose of 1 mg/kg. The application in either preparation was well tolerated. In seven dogs, peak levels in the range of 730 ng/mL to 1,000 ng/mL were measured after intravenous injection with an area under curve (AUC) of 75 ng.h/kg.mL to 146 ng.h/kg.mL. It was of note that even the oral administration of the same busulfan preparation resulted in AUC values in the same range as observed after parenteral application. The absorption rate of busulfan tablets in our model was as unpredictable as documented in clinical trials. On the basis of the present study, clinical trials using busulfan dissolved in DMSO given either intravenously or orally appear warranted. This approach should lead to predictable blood levels, reduced toxicity, and increased efficacy of busulfan-containing regimens.
ALL-1 gene rearrangements in DNA topoisomerase II inhibitor-related leukemia in children
Authors: Felix CA , Hosler MR , Winick NJ , Masterson M , Wilson AE , Lange BJ
Published: October 2019

We examined clinical, morphologic, and cytogenetic features and ALL-1 (MLL, Htrxl, HRX) gene rearrangements in 17 cases of secondary leukemia that occurred 11 months to 9 years from diagnoses of primary cancers in children who received topoisomerase II inhibitors or developed secondary leukemias typical of those associated with this therapy. Primary diagnoses included nine solid tumors and eight leukemias. Ten secondary leukemias were acute myeloid leukemia (AML), one was of mixed lineage, two were acute lymphoblastic leukemia (ALL), and four presented as myelodysplasia. Of 15 cases with 11q23 involvement, 11 (73%) were cytogenetically identifiable; four cases had molecular rearrangement only. By Southern blot, rearrangements within the ALL-1 gene were similar to sporadic cases. The results of this analysis suggest the following: (1) In most pediatric cases of topoisomerase II inhibitor-associated leukemia, there is disruption of the breakpoint cluster region of the ALL-1 gene at chromosomal band 11q23. (2) Exposure histories vary in secondary 11q23 leukemia, as the only topoisomerase II inhibitor was dactinomycin in one case, and, in another case, no topoisomerase II inhibitor was administered. (3) There is clinical, morphologic, cytogenetic, and molecular heterogeneity in pediatric secondary 11q23 leukemia. (4) There are some survivors of pediatric secondary 11q23 leukemia, but the outcome is most often fatal.
Percentage of Philadelphia chromosome (Ph)-negative and Ph-positive cells found after autologous transplantation for chronic myelogenous leukemia depends on percentage of diploid cells induced by conventional- dose chemotherapy before collection of autologous cells
Authors: Talpaz M , Kantarjian H , Liang J , Calvert L , Hamer J , Tibbits P , Durett A , Claxton D , Giralt S , Khouri I
Published: October 2019

We collected peripheral blood mononuclear cells and bone marrow cells soon after recovery from conventional-dose chemotherapy-induced myelosuppression and transplanted these cells into advanced chronic myelogenous leukemia (CML) patients after treating these patients with 120 mg/kg cyclophosphamide, 750 mg/m2 VP-16, and 1,020 cGy of total body irradiation (TBI). Of the 10 late chronic-phase patients and the eight accelerated-phase CML patients evaluable posttransplant, 90% and 87%, respectively, remain alive posttransplant, whereas none of the three blast crisis CML patients given this therapy remain alive posttransplant. We measured the percentage of Philadelphia chromosome (Ph)-negative cells in the autologous cells collected after conventional-dose chemotherapy-induced myelosuppression before autologous transplant and in the marrow of these same CML patients after autologous transplantation of these cells into recipients treated with the cyclophosphamide, VP-16, and TBI. A direct correlation (correlation coefficient = 0.91) was observed between the level of Ph+ cells in the transplanted cells and the percentage of Ph+ marrow cells after transplant in 21 patients so transplanted. The data show that the chance of generating cytogenetic remissions post-transplant depends on the percentage of diploid cells in the preparations of autologous cells used for transplant and the stage of disease of the patients at the time of collection of the autologous cells.
Impact of magnetic resonance imaging on the diagnosis of abdominal complications of paroxysmal nocturnal hemoglobinuria
Authors: Mathieu D , Rahmouni A , Villeneuve P , Anglade MC , Rochant H , Vasile N
Published: October 2019

Magnetic resonance (MR) imaging is a method of choice for assessing vascular patency and parenchymal iron overload. During the course of paroxysmal nocturnal hemoglobinuria (PNH), it is clinically relevant to differentiate abdominal vein thrombosis from hemolytic attacks. Furthermore, the study of the parenchymal MR signal intensity adds informations about the iron storage in kidneys, liver, and spleen. Twelve PNH patients had 14 MR examinations of the abdomen with spin-echo T1- and T2-weighted images and flow-sensitive gradient echo images. Vessels patency and parenchymal signal abnormalities--either focal or diffuse--were assessed. MR imaging showed acute complications including hepatic vein obstruction in five patients, portal vein thrombosis in two patients, splenic infarct in one patient. In one patient treated with androgens, hepatocellular adenomas were shown. Parenchymal iron overload was present in the renal cortex of eleven patients with previous hemolytic attacks. On the first MR study of the remaining patient with an acute abdominal pain showing PNH, no iron overload was present in the renal cortex. Follow-up MR imaging showed the onset of renal cortex iron overload related to multiple hemolytic attacks. Despite the fact that all our patients were transfused, normal signal intensity of both liver and spleen was observed in three of them. MR imaging is particularly helpful for the diagnosis of abdominal complications of PNH.
Antisickling effects of 2,3-diphosphoglycerate depletion
Authors: Poillon WN , Kim BC , Labotka RJ , Hicks CU , Kark JA
Published: October 2019

Elevation of 2,3-bisphosphoglycerate (2,3-DPG) in sickle erthrocytes (SS RBCs) and concomitant acidification of the cell interior promote polymerization by decreasing the solubility (csat) of deoxyhemoglobin S. The antisickling effect of 2,3-DPG depletion was evaluated after activation of the 2,3-DPG phosphatase activity of bisphosphoglycerate mutase by glycolate-2-phosphate, leading to rapid loss of intracellular 2,3-DPG. To ensure its maximal reduction in a physiologic medium, isosmotic CO2/bicarbonate-buffered saline, pH 7.0, was used. Substitution of K+ for Na+ as the major extracellular cation suppressed K:Cl cotransport, prevented cell shrinkage, and allowed demonstration of the full antisickling effect of 2,3-DPG depletion. The modest effect on solubility per se of removing intraerythrocytic 2,3-DPG (delta Csat = 1.6 g/dL) was amplified into a much larger antisickling effect by interaction with three other cellular variables affecting solubility and polymer content (intracellular pH, O2 saturation, and mean cell hemoglobin concentration). Acting in concert, these four antisickling effects (three solubilizing, one osmotic) reduced polymer fraction of glycolate-treated SS RBCs by 32% to 63%, with a concomitant decrease in sickling of 46% to 95% at the nominal pO2 of the microcirculation (20 mm Hg). A decrement in sickling of this magnitude should significantly ameliorate the vasoocclusive severity of sickle cell disease.
Expression of adhesion molecules on CD34+ cells: CD34+ L-selectin+ cells predict a rapid platelet recovery after peripheral blood stem cell transplantation
Authors: Dercksen MW , Gerritsen WR , Rodenhuis S , Dirkson MK , Slaper-Cortenbach IC , Schaasberg WP , Pinedo HM , von dem Borne AE , van der Schoot CE
Published: October 2019

Adhesion molecules play a role in the migration of hematopoietic progenitor cells and regulation of hematopoiesis. To study whether the mobilization process is associated with changes in expression of adhesion molecules, the expression of CD31, CD44, L-selectin, sialyl Lewisx, beta 1 integrins very late antigen 4 (VLA-4) and VLA-5, and beta 2 integrins lymphocyte function-associated 1 and Mac-1 was measured on either bone marrow (BM) CD34+ cells or on peripheral blood CD34+ cells mobilized with a combination of granulocyte colony-stimulating factor (G-CSF) and chemotherapy. beta 1 integrin VLA-4 was expressed at a significantly lower concentration on peripheral blood progenitor cells than on BM CD34+ cells, procured either during steady-state hematopoiesis or at the time of leukocytapheresis. No differences in the level of expression were found for the other adhesion molecules. To obtain insight in which adhesion molecules may participate in the homing of peripheral blood stem cells (PBSCs), the number of CD34+ cells expressing these adhesion molecules present in leukocytapheresis material was quantified and correlated with hematopoietic recovery after intensive chemotherapy in 27 patients. The number of CD34+ cells in the subset defined by L-selectin expression correlated significantly better with time to platelet recovery after PBSC transplantation (r = -.86) than did the total number of CD34+ cells (r = -.55). Statistical analysis of the relationship between the number of CD34+L-selectin+ cells and platelet recovery resulted in a threshold value for rapid platelet recovery of 2.1 x 10(6) CD34+ L-selectin+ cells/kg. A rapid platelet recovery (< or = 14 days) was observed in 13 of 15 patients who received > or = 2.1 x 10(6) CD34+ L-selectin+ cells/kg (median, 11 days; range, 7 to 16 days), whereas 10 of 12 patients who received less double positive cells had a relative slow platelet recovery (median, 20 days; range, 13 to 37 days). The L-selectin+ subpopulation of CD34+ cells also correlated better with time to neutrophil recovery (r = -.70) than did the total number of reinfused CD34+ cells (r = -.51). However, this latter difference failed to reach statistical significance. This study suggests that L-selectin is involved in the homing of CD34+ cells after PBSC transplantation.
Tumor necrosis factor ligand superfamily: involvement in the pathology of malignant lymphomas
Authors: Gruss HJ , Dower SK
Published: October 2019

The TNF receptor superfamily members are all type I membrane glycoproteins with typical homology in the extracellular domain of variable numbers of cysteine-rich repeats (overall homologies, 25% to 30%). In contrast, the TNF ligand superfamily members (with the exception of LT alpha) are type II membrane glycoproteins with homology to TNF in the extracellular domain (overall homologies, 20%). TNF and LT alpha are trimeric proteins and are composed of beta-strands forming a beta-jellyroll. The homology of the beta-strand regions for the TNF ligand superfamily members suggest a similar beta-sandwich structure and possible trimeric or multimeric complex formation for most or all members. A genetic linkage, as evidence for evolutionary relatedness, is found by chromosomal cluster of TNFR p80, CD30, 4–1BB, and OX40 for 1p36; TNFR p60, TNFR-RP, and CD27 for 12p13; TNF, LT alpha, and LT beta for 6 (MHC locus); CD27L and 4–1BBL for 19p13; and FASL and OX40L for 1q25. Of the TNF ligand superfamily, TNF, LT alpha, and LT beta and their receptors (TNFR p60, TNFR p80, and TNFR-RP) interact in a complex fashion of cross-binding. However, the other family members presently have a one ligand/one receptor binding principle (CD27/CD27L, CD30/CD30L, CD40/CD40L, 4–1BB/4–1BBL, OX40/gp34, and FAS/FASL). In general, the members of the TNF ligand superfamily mediate interaction between different hematopoietic cells, such as T cell/B cell, T cell/monocyte, and T cell/T cell. Signals can be transduced not only through the receptors but also through at least some of the ligands. The transduced signals can be stimulatory or inhibitory depending on the target cell or the activation state. Taken together, TNF superfamily ligands show for the immune response an involvement in the induction of cytokine secretion and the upregulation of adhesion molecules, activation antigens, and costimulatory proteins, all known to amplify stimulatory and regulatory signals. On the other hand, differences in the distribution, kinetics of induction, and requirements for induction support a defined role for each of the ligands for T-cell-mediated immune responses. The shedding of members of the TNF receptor superfamily could limit the signals mediated by the corresponding ligands as a functional regulatory mechanism. Induction of cytotoxic cell death, observed for TNF, LT alpha, CD30L, CD95L, and 4–1BBL, is another common functional feature of this cytokine family. Further studies have to identify unique versus redundant biologic and physiologic functions for each of the TNF superfamily ligands.(ABSTRACT TRUNCATED AT 400 WORDS)
p15ink4B and p16ink4 gene inactivation in acute lymphocytic leukemia
Authors: Rasool O , Heyman M , Brandter LB , Liu Y , Grander D , Soderhall S , Einhorn S
Published: October 2019

Malignant cells from 52 children with acute lymphocytic leukemia (ALL) were investigated for inactivation of the p15ink4B and p16ink4 genes and other genetic alterations on chromosome 9p21. Homozygous deletions of the p15ink4B and/or the p16ink4 genes were detected in 16 cases and a further 9 cases showed evidence of allelic loss either by hemizygous deletion or loss of heterozygosity (LOH) for 9p21 markers. Most cases had loss of both genes, but 5 patients had lost only p16ink4 and 2 cases had homozygous loss of p15ink4B only. Sequence analysis of all exons of p15ink4B and p16ink4 was performed in patients with hemizygous deletions or LOH for 9p21 markers. A frame shift mutation of p16ink4 exon 1 was shown in 1 case, whereas all other clones carried the wild-type sequence of p15ink4B and p16ink4 in the remaining allele. The data suggest that both the p15ink4B and p16ink4 genes can be inactivated in ALL. The existence of a hitherto undefined tumor-suppressor gene on chromosome 9p cannot be ruled out.
Inhibition of endotoxin-induced activation of the coagulation and fibrinolytic pathways using a recombinant endotoxin-binding protein (rBPI23)
Authors: von der Mohlen MA , van Deventer SJ , Levi M , van den Ende B , Wedel NI , Nelson BJ , Friedmann N , ten Cate JW
Published: October 2019

A recombinant endotoxin-neutralizing protein, rBPI23, was shown to partially prevent endotoxin-induced activation of the fibrinolytic and coagulation systems in experimental endotoxemia in humans. In a placebo-controlled, blinded crossover study, eight volunteers were challenged twice with an intravenous bolus injection of endotoxin (40 EU/kg of body weight) and concurrently received either rBPI23 (1 mg/kg) or placebo (human serum albumin, 0.2 mg/kg). rBPI23 treatment significantly lowered the endotoxin-induced fibrinolytic response, ie, reduced the release of tissue-type plasminogen activator, urokinase-type plasminogen activator, plasminogen activator inhibitor antigen, and complex formation of plasmin alpha 2-antiplasmin (P = .0078 for each). Plasminogen activator inhibitor activity was also reduced, but not significantly according to the Hochberg method (P = .0304). The endotoxin-induced activation of the procoagulant state as reflected by increase in F1 + 2 fragments and TAT complexes was blunted by rBPI23 infusion (P = .0391 [not significant according to the Hochberg method] and .0078, respectively). These results indicate that rBPI23 is capable of reducing both the activation of the fibrinolytic and the coagulation systems after low-dose endotoxin infusion in humans.
Continuous infusion of the anti-CD22 immunotoxin IgG-RFB4-SMPT-dgA in patients with B-cell lymphoma: a phase I study
Authors: Sausville EA , Headlee D , Stetler-Stevenson M , Jaffe ES , Solomon D , Figg WD , Herdt J , Kopp WC , Rager H , Steinberg SM
Published: October 2019

IgG-RFB4-SMPT-dgA consists of deglycosylated ricin A chain (dgA) coupled to the monoclonal antihuman CD22 antibody, RFB4. This study determined the maximally tolerated dose (MTD) of this immunotoxin (IT) administered as a continuous 8-day infusion to 18 patients with B-cell lymphoma (30% CD22+ tumor cells) over 8 days. The MTD was 19.2 mg/m2/192 h (maximum toxicity grade 1), with vascular leak syndrome (VLS) as dose-limiting toxicity (DLT) at 28.8 mg/m2/192 h (grades 3 through 5 in 7 of 11 patients). Predictors of severe VLS included serum IT concentrations greater than 1,000 ng/mL and the absence of circulating tumor cells. Decreased urine sodium excreted in 24 hours provided evidence for mild VLS without notable changes in serum albumin. Four partial responses, 3 minor responses, 6 stable disease, and 3 progression of disease were observed. The mean maximal serum concentration (Cmax) in initial courses at the MTD (19.2 mg/m2) was 443 +/- 144 ng/mL (n = 3; range, 326 to 604). At 28.8 mg/m2/192 h, the Cmax was highly variable (n = 11; mean, 1,102 +/- 702; range, 9.6 to 2,032 ng/mL). Human antimouse or antiricin antibodies developed in 6 of 16 (37.5%) patients after one course of IT. However, 10 eligible patients received multiple courses of IT. Changes in serum cytokines and cytokine receptors did not correlate with toxicity but decreased soluble interleukin-2 receptor concentrations correlated with clinical response. Comparison to a prior study with the same IT administered by intermittent bolus infusions (Amlot et al, Blood 82:2624, 1993) suggests similar clinical response, toxicity, and immunogenicity.
Interleukin-13 selectively suppresses the growth of human macrophage progenitors at the late stage
Authors: Sakamoto O , Hashiyama M , Minty A , Ando M , Suda T
Published: October 2019

Interleukin-13 (IL-13) is a pleiotropic cytokine that inhibits the production of inflammatory cytokines of monocytes. We investigated the effects of IL-13 on the clonal growth of human hematopoietic progenitors. IL-13 alone did not support any colony formation. IL-13 markedly suppressed macrophage colonies that were formed in the presence of IL-3 and erythropoietin, granulocyte-macrophage colony-stimulating factor, or macrophage colony-stimulating factor. Macrophage colony cells showed dendritic cell-line morphology and cellular aggregates. IL-13 did not affect granulocyte colony and erythroid burst formation. Delayed addition of IL-13 and replating onto the culture dishes with IL-13 showed that macrophage colony formation was suppressed during days 8 and 14 of culture. These results indicate that IL-13 affects the growth of the late stage of committed macrophage progenitors. Single-cell culture of isolated CD34+CD33+ cells with IL-13 confirmed that macrophage colony formation was significantly suppressed. These results show that IL-13 directly suppresses the proliferation of differentiating macrophages. In addition, these suppressive effects of IL-13 were synergistic with IL-4. Furthermore, in the liquid culture of bone marrow cells in the presence of IL-13, the number of CD14 (monocyte-macrophage antigen)-positive cells decreased and CD18 (LFA-1 beta)-positive cells increased. It is concluded that IL-13 affects the growth of the late stage of macrophage precursors as well as mature monocytes. Induction of differentiation of human monocytes may be correlated with the suppression of their progenitors.
Autocrine interleukin-1 beta regulates both proliferation and apoptosis in EL4-6.1 thymoma cells
Authors: Fratelli M , Gagliardini V , Galli G , Gnocchi P , Ghiara P , Ghezzi P
Published: October 2019

We demonstrate here that EL4–6.1 cells, a mouse thymoma that expresses high levels of membrane interleukin (IL)-1 receptors, produce IL-1 beta as an autocrine regulatory factor. Endogenous IL-1 beta sustains both proliferation and apoptosis: during the exponential phase, it mainly promotes proliferation, while during the plateau phase of cell growth, it induces death by apoptosis. Additionally, we show that exogenous IL-1 beta added to EL4–6.1 cells in lag phase induces apoptosis in a portion of the cells and proliferation in the remaining cells. Therefore, IL-1 beta can exert two completely opposite effects on a single cell type, depending on the state of the target cell.
Expression and function of Fas (APO-1/CD95) in patient myeloma cells and myeloma cell lines
Authors: Westendorf JJ , Lammert JM , Jelinek DF
Published: October 2019

Cross-linkage of the Fas antigen induces programmed cell death in many normal and malignant lymphoid cells by a process known as apoptosis. In this study, we examined the sensitivity of myeloma cell lines and patient plasma cells to a cytolytic anti-Fas monoclonal antibody (MoAb). Eight of 10 myeloma cell lines were induced to undergo programmed cell death by anti-Fas MoAb as determined by DNA fragmentation and morphologic changes. Of the two myeloma cell lines that were resistant to anti-Fas treatment, one did not express the Fas antigen. Only the U266 cell line expressed Fas, but was not killed by the anti0Fas MoAb. To extend these studies, we have examined the expression and function of Fas in freshly isolated CD38hiCD45neg-int plasma cells from patients with multiple myeloma (MM), monoclonal gammopathy of undetermined significance (MGUS), and primary amyloidosis (AL). By three-color flow cytometry, we found Fas expression in CD38hiCD45neg-int plasma cells from all patient groups to be variable, as Fas was expressed in 15 of 28 MM, 3 of 6 MGUS, and 2 of 7 AL patients. In morphologic studies of apoptosis, Fas-positive myeloma cells in patient bone marrow mononuclear cell (MNC) cultures appeared to be resistant to anti-Fas-mediated apoptosis. By contrast, purified myeloma cells from the same patient were sensitive to anti-Fas treatment, suggesting the presence of a protective factor(s) in unseparated MNC cultures that may inhibit Fas-induced apoptosis of plasma cells. Of interest, serum from normal individuals and myeloma patients also protected myeloma cell lines from undergoing Fas-mediated apoptosis. These studies show that Fas expression in myeloma cell lines and CD38hiCD45neg-int patient plasma cells is variable and may reflect a variance in the maturation status of the various plasma cell populations. Moreover, Fas-mediated killing of patient cells and myeloma cell lines was also variable, which may be influenced, in part, by the presence of a soluble protective factor.
Early human thymocyte proliferation is regulated by an externally controlled autocrine transforming growth factor-beta 1 mechanism
Authors: Mossalayi MD , Mentz F , Ouaaz F , Dalloul AH , Blanc C , Debre P , Ruscetti FW
Published: October 2019

Early thymocytes undergo extensive proliferation after their entry into the thymus, but cellular interactions and cytokines regulating this intrathymic step remain to be determined. We analyzed the effects of various T-cell growth factors and cellular interactions on in vitro proliferation of early CD2+CD3/TCR-CD4-CD8-(triple negative [TN]) human thymocytes. Freshly isolated TN cells were then assayed for their growth capacity after incubation with CD2I+III-monoclonal antibody (MoAb), recombinant human interleukin-2 (IL-2), IL-7, and/or IL-4. These cells displayed significant proliferative responses with IL-4, IL-7, or CD2-MoAb+IL-2. The addition of recombinant transforming growth factor beta (TGF beta) or autologous irradiated CD3+CD8+CD4- cells to TN cell cultures dramatically decreased their growth responses to IL-2 and IL-7, whereas IL-4-induced proliferation was less sensitive to growth inhibition. We thus asked whether the CD8+ cell-derived inhibitory effect was due to TGF beta. The addition of neutralizing anti-TGF beta MoAb completely abolished CD8+ cell-derived inhibition of TN cell growth. Analysis of CD8+ cell-derived supernatants indicated that these cells had low TGF beta 1 production capacity, whereas TN cells secrete significantly high levels of TGF beta 1. Cell fixation studies showed that TN cells were the source of the TGF beta. TGF beta 1 released from TN cells was in the latent form that became the active inhibitory form through interaction of TN cells with CD8+ cells. Together, these data suggest a role for TGF beta 1 as an externally controlled, autocrine inhibitory factor for human early thymocytes, with a regulatory role in thymic T-cell output.
Cytogenetic profile of minimally differentiated (FAB M0) acute myeloid leukemia: correlation with clinicobiologic findings [see comments]
Authors: Cuneo A , Ferrant A , Michaux JL , Boogaerts M , Demuynck H , Van Orshoven A , Criel A , Stul M , Dal Cin P , Hernandez J
Published: October 2019

Cytogenetic data were studied in 26 patients with de novo acute myeloid leukemia (AML) with minimal myeloid differentiation, corresponding to the M0 subtype of the French-American-British classification, in correlation with cytoimmunologic and clinical findings. Clonal abnormalities were detected in 21 cases (80.7%), 12 of which had a complex karyotype. Partial or total monosomy 5q and/or 7q was found, either as the sole aberration or in all abnormal metaphases, in 11 patients; in 8 cases, additional chromosome changes were present, including rearrangements involving 12p12–13 and 2p12–15 seen in 3 cases each. Five patients had trisomy 13 as a possible primary chromosome change; in 5 cases, nonrecurrent chromsome abnormalities were observed. Comparison of these findings with chromosome data from 42 patients with AML-M1 shows that abnormal karyotypes, complex karyotypes, unbalanced chromosome changes (-5/5q- and/or -7/7q- and +13) were observed much more frequently in AML-M0 than in AML-M1. Patients with abnormalities of chromosome 5 and/or 7 frequently showed trilineage myelodysplasia and low white blood cell count. Despite their relatively young age, complete remission was achieved in 4 of 11 patients only. Patients with +13 were elderly males with frequent professional exposure to myelotoxic agents. Unlike patients with clonal abnormalities, most AML-M0 patients with normal karyotype showed 1% to 2% peroxidase-positive blast cells at light microscopy and frequently achieved CR. It is concluded that (1) AML-M0 shows a distinct cytogenetic profile, partially recalling that of therapy-related AML, (2) different cytogenetic groups of AML-M0 can be identified showing characteristic clinicobiologic features, and (3) chromosome rearrangements may partially account for the unfavorable outcome frequently observed in these patients.
Expression of CD21 antigen on myeloma cells and its involvement in their adhesion to bone marrow stromal cells
Authors: Huang N , Kawano MM , Mahmoud MS , Mihara K , Tsujimoto T , Niwa O , Kuramoto A
Published: October 2019

The mature myeloma cells express very late antigen 5 (VLA-5) and MPC-1 antigens on their surface and adhere to bone marrow (BM) stromal cells more tightly than the VLA-5-MPC-1-immature myeloma cells in vitro. The VLA-5 and MPC-1 antigens possibly function as two of the molecules responsible for interaction of mature myeloma cells with BM stromal cells. However, the immature myeloma cells do interact with BM stromal cells, and it is unclear which adhesion molecules mediate their interaction. In this study, we found that both immature and mature myeloma cells expressed CD21, an adhesion molecule known to bind to CD23. CD21 was also detected on normal plasma cells. To evaluate the role of CD21 expression on myeloma cells, two myeloma cell lines, NOP-2 (VLA-5-MPC-1-) and KMS-5 (VLA-5+MPC-1+), were used as representatives of immature and mature myeloma cell types, respectively, and an adhesion assay was performed between the myeloma cell lines and BM stromal cells. Antibody-blocking results showed that adhesion of the mature type KMS-5 to KM102, a human BM-derived stromal cell line, or to short-term cultured BM primary stromal cells was inhibited by monoclonal antibodies (MoAbs) against CD21, VLA-5, and MPC-1, and inhibition of adhesion of the immature type NOP-2 to KM102 by the anti-CD21 MoAb was observed as well. Furthermore, CD23 was detected on KM102. Treatment of KM102 with an anti-CD23 MoAb also inhibited adhesion of either KMS-5 or NOP-2 to KM102. Therefore, we propose that CD21 expressed on myeloma cells likely functions as a molecule responsible for the interaction of immature myeloma cells as well as mature myeloma cells with BM stromal cells, and CD23 may be the ligand on the stromal cells for the CD21-mediated adhesion.
Sialylated O-glycans and L-selectin sequentially mediate myeloid cell rolling in vivo
Authors: Ley K , Zakrzewicz A , Hanski C , Stoolman LM , Kansas GS
Published: October 2019

Leukocyte rolling precedes firm adhesion and emigration in inflammatory cell recruitment. Both P-selectin, an endothelial lectin that binds to sialylated O-glycans containing sialyl-Lewisx (sLex) on the granulocyte surface, and leukocyte L-selectin have been shown to mediate leukocyte rolling in vivo. Here, we investigate rolling of isolated human neutrophils (PMN), HL-60 promyelocytes, and an L-selectin-transfected cell line (300.19-L) during trauma-induced inflammation in rat mesenteric venules. HL-60 cells, which express no L-selectin but abundant sLex, rolled effectively immediately after abdominal surgery. HL-60 cell rolling was almost completely abolished by pretreatment with sialidase or monoclonal antibody (MoAb) AM-3 recognizing sLex, and was reduced by about 80% by O-sialoglycoprotein-endopeptidase (OSGP). By contrast, 300.19-L cells rolled poorly immediately after surgery but rolled well between 40 and 120 minutes after surgery. Their rolling was completely inhibited by the blocking L-selectin MoAb LAM1–3, but not by a binding control MoAb. PMN express both L-selectin and clustered, sialylated glycoproteins including P-selectin glycoprotein ligand-1 (PSGL-1). PMN showed effective rolling at all times, which was abolished by sialidase or MoAb AM-3 pretreatment during the first 30 minutes after surgery, but not later, when PMN rolling was largely L-selectin-dependent. We conclude that in trauma-induced inflammation, a two-step mechanism accounts for most of myeloid cell rolling, which initially requires O-glycans and subsequently depends on L-selectin function.
The coding sequence of Duffy blood group gene in humans and simians: restriction fragment length polymorphism, antibody and malarial parasite specificities, and expression in nonerythroid tissues in Duffy- negative individuals
Authors: Chaudhuri A , Polyakova J , Zbrzezna V , Pogo AO
Published: October 2019

The coding and untranslated flanking sequences of Duffy gene (FY) in humans and simians are in a single exon. The difference between the two codominant alleles, FY*A and FY*B, is a single change at nucleotide 306: guanidine is in FY*A and adenine is in FY*B. This produces a codon change that subsequently modifies the amino acid at position 43 of gpFy, the major subunit of the Duffy blood group protein complex. The glycine at this position in antigen Fya exchanges with aspartic acid in antigen Fyb. The guanidine at nucleotide 306 creates an additional Ban I restriction site in FY*A. Ban I digestion of DNA-PCR amplified products of FY*B and FY*A yields three and four fragments, respectively. Restriction fragment length polymorphism (RFLP) studies show that Fy(a+b-) and Fy(a-b+) whites are FY homozygous, that most Fy(a-b-) blacks have FY*B, and most Fy(a+b-) blacks are FY*A/FY*B heterozygous. In the black population a silent FY*B is very common, but a silent FY*A has not been found yet. On RNA blot analysis, the gpFy cDNA clone detected mRNA in the lung, spleen, and colon but not in the bone marrow of Duffy-negative individuals. Therefore, there is no null phenotype in Fy(a-b-) blacks. The gpFy homology between human and chimpanzee is 99% with a single residue change at position 116 (valine to isoleucine), whereas a 94% homology is found in squirrel and rhesus monkeys, and there is a 93% homology in aotus monkey when compared with humans. The N-terminal exocellular domain of simian gpFy helps to identify a set of amino acids critical for antibody and malarial parasite specificities.
Secretion of cytokines (interleukins-1 alpha, -3, and -6 and granulocyte-macrophage colony-stimulating factor) by normal human bone marrow megakaryocytes
Authors: Wickenhauser C , Lorenzen J , Thiele J , Hillienhof A , Jungheim K , Schmitz B , Hansmann ML , Fischer R
Published: October 2019

The effects of cytokine stimulation [recombinant human interleukin (rhIL)-1 alpha, rhIL-3, rhIL-6, rhIL-11, and rh granulocyte-macrophage colony-stimulating factor (GM-CSF)] on the secretory activity of normal human megakaryocytes were studied by means of the reverse hemolytic plaque assay (RHPA) in enriched cell preparations. This test facilitates an extremely sensitive determination of cytokine secretion at the single-cell level, together with the clear-cut identification of each immunostained (CD61) secretory active megakaryocyte. Moreover, the reverse transcriptase-polymerase chain reaction (RT-PCR) was used to investigate the expression of IL-6, IL-6 receptor (IL-6R), IL-9, IL-10, IL-12, and IL-13 mRNA in highly concentrated megakaryocyte preparations. In comparison with the spontaneous secretion rate, stimulation with rhIL-1 alpha, rhIL-6, and rhGM-CSF failed to induce a significant increase in the release of cytokines by CD61+ cells. On the other hand, both rhIL-3 and, in a less pronounced way, rhIL-11 exerted a marked effect on IL-6 secretion. Additionally, after stimulation with rhIL-3, a significant enhancement of the secretion of IL-3 and GM-CSF, but not of IL-1 alpha, could be observed. Using the RT-PCR, a significant induction of IL-6 expression could be appreciated in the enriched megakaryocyte population (60% to 80%) stimulated with rhIL-3. The results of this study provide persuasive evidence that a number of cytokines are synthesized and secreted by human megakaryocytes and not only by hematopoietic stroma cells. These data suggest the existence of autocrine and paracrine mechanisms that may influence maturation and differentiation of megakaryocytes as well as act on various stroma cells to sustain an appropriate hematopoietic micro-environment.
Calin from Hirudo medicinalis, an inhibitor of platelet adhesion to collagen, prevents platelet-rich thrombosis in hamsters
Authors: Deckmyn H , Stassen JM , Vreys I , Van Houtte E , Sawyer RT , Vermylen J
Published: October 2019

Interaction between exposed collagen and platelets and/or von Willebrand factor is believed to be one of the initiating events for thrombus formation at sites of damaged endothelium. Interference with this mechanism may provide an anti-thrombotic potential. Calin, a product from the saliva of the leech Hirudo medicinalis, was tested in vitro and for its in vivo activity in a thrombosis model in hamsters. Calin specifically and dose dependently (IC50:6.5 to 13 micrograms/mL) inhibited human platelet aggregation induced by collagen. In addition, specific platelet adhesion onto microtiter wells coated with collagen and detected with a monoclonal antiglycoprotein IIb/IIIa antibody- conjugated with horseradish peroxidase, could be completely prevented with Calin (IC50:22 micrograms/mL). A dose-response curve was constructed in groups of six hamsters in whom a standardized trauma was induced on the femoral vein. Thrombus formation was followed continuously using video recording and processing of the image obtained upon transillumination of the vessel. Intravenous Calin dose- dependently inhibited platelet-rich thrombus formation in this model with an ED50 of 0.07 mg/kg and complete inhibition with 0.2 mg/kg. No effects were seen on coagulation tests or bleeding times, whereas ex vivo aggregation induced by collagen was inhibited dose dependently. Local application of leech saliva, Calin, hirudin, or the combination of the latter two into the bleeding time wound of hamsters resulted in a mild prolongation of the bleeding time (twofold to threefold). A similar experiment in baboons did not cause any prolongation of the bleeding time. This is in sharp contrast with the long-lasting bleeding after a leech bite itself in both species. Calin from the leech Hirudo medicinalis is able, by binding to collagen, to effectively interfere with platelet-collagen interaction, which results in an antithrombotic effect observed in a platelet-rich thrombosis model in hamsters.
Fibrinogen inhibits the heparin cofactor II-mediated antithrombin activity of dermatan sulfate
Authors: Zammit A , Dawes J
Published: October 2019

Dermatan sulfate is a naturally occurring antithrombotic glycosaminoglycan. The antithrombin activity of several dermatan sulfate preparations has been measured in whole human plasma and found to be -55% of that in purified systems. Kinetic studies under pseudo- first-order conditions indicated that the reduction in antithrombin activity of dermatan sulfate in plasma compared with that in buffer was due to noncompetitive inhibition with respect to dermatan sulfate. Analysis of the protein profile bound to immobilized dermatan sulphate showed that on a molar basis, histidine-rich glycoprotein and apolipoprotein E were the most abundant proteins specifically bound, together with significant amounts of fibrinogen and vitronectin. Addition of these proteins to the purified system showed that only fibrinogen inhibited the antithrombin activity of dermatan sulfate and that it did so in a concentration-dependent manner over the physiologic range of plasma fibrinogen levels. These results indicate that the anticoagulant activity of dermatan sulfate may be modulated in human plasma by fibrinogen.
Immunoreactive interleukin-6 and acute phase proteins as prognostic factors in multiple myeloma. Finnish Leukemia Group
Authors: Pelliniemi TT , Irjala K , Mattila K , Pulkki K , Rajamaki A , Tienhaara A , Laakso M , Lahtinen R
Published: October 2019

High serum level of bioactive interleukin-6 (IL-6) is regarded as a predictor of poor prognosis in multiple myeloma (MM). On the other hand, the reported levels of immunoreactive IL-6 have been highly variable, and the prognostic value of immunoreactive IL-6 in MM is not clear. We have analyzed the prognostic significance of serum immunoreactive IL-6, as measured by a sensitive immunosorbent assay, in 210 patients with newly diagnosed MM subsequently treated with intermittent melphalan and prednisone. The serum levels of acute phase proteins C-reactive protein (CRP), alpha 1-antitrypsin (alpha 1AT), and acid alpha 1-glycoprotein (orosomucoid; OM) were evaluated as surrogates for IL-6. Serum IL-6, CRP, alpha 1AT, and OM levels were raised in 42%, 40%, 41%, and 24% of the patients, respectively. There was a significant correlation between the clinical stage of the patients and serum IL-6 (P = .006), alpha 1AT (P = .001), and OM (P = .004) levels at diagnosis. At 3 years, 52% of the patients were alive. Univariate logistic regression analysis showed that high levels of IL-6 (P = .002), CRP (P = .02), alpha 1AT (P < .001), OM (P = .007), beta 2- microglobulin (beta 2M; P < .001), and thymidine kinase (P < .05) were all associated with 3-year mortality. In multivariate regression analysis, beta 2M (P < .0001) and alpha 1AT (P = .01) had independent prognostic significance. The patients with high levels of both beta 2M and alpha 1AT or IL-6 were at very high risk of dying within 3 years from diagnosis (16% and 21% of the patients in these groups were alive, respectively). When the patients were stratified according to the clinical stage, the prognostic significance of serum IL-6 and alpha 1AT was especially evident in stage II patients. When the patients were divided into two groups according to normal or raised serum IL-6 levels, the patients with high IL-6 levels had more frequent osteolytic bone lesions (P = .03) and a more aggressive disease. We conclude that serum immunoreactive IL-6 is a significant prognostic marker in MM.
Granulocyte colony-stimulating factor induction of normal human bone marrow progenitors results in neutrophil-specific gene expression
Authors: Berliner N , Hsing A , Graubert T , Sigurdsson F , Zain M , Bruno E , Hoffman R
Published: October 2019

We have used a combination of hematopoietic growth factors to induce in vitro granulocytic maturation. A fraction of marrow cells enriched for hematopoietic progenitor cells (CD34+, HLA-DR+) was isolated from normal human bone marrow by monoclonal antibody staining and fluorescence-activated cell sorting. Cells were cultured in a suspension system for 3 days in the presence of stem cell factor and interleukin-3 (IL-3), after which granulocyte colony-stimulating factor (G-CSF) was added. Cells were harvested daily and analyzed for phenotypic maturation by morphologic criteria, and total RNA was obtained for analysis of myeloid gene expression. Maturation was observed to progress to the late metamyelocyte and band stage over a period of 10 to 12 days. Neutrophil-specific gene expression was assayed by reverse transcription-polymerase chain reaction (RT-PCR). Induction with G-CSF resulted in sequential expression of primary and secondary granule proteins, with asynchronous expression of primary granule proteins starting from days 1 to 5, and synchronous expression of lactoferrin and transcobalamin I (secondary granule proteins) from days 7 to 8. Interestingly, myeloperoxidase (MPO) mRNA expression was easily detected in both the freshly isolated CD34+, HLA-DR+ cells and cells at all subsequent stages of induction. This suggests that MPO mRNA is expressed very early during neutrophil development, perhaps before the development of significant numbers of phenotypically recognizable granules. This recapitulation of a program of sequential expression of primary and secondary granule protein genes suggests that in vitro marrow culture suspensions to which appropriate growth factors are added can mimic normal granulocytic maturation. This system should provide an important model for the study of neutrophil-specific gene expression.