bluuebunny/crossref_metadata_2025 huggingface dataset:
Human erythrocyte band 3 polymorphism (band 3 Memphis): characterization of the structural modification (Lys 56----Glu) by protein chemistry methods
Authors: Yannoukakos D , Vasseur C , Driancourt C , Blouquit Y , Delaunay J , Wajcman H , Bursaux E
Published: October 2019

Band 3 variants occur rather frequently in different populations. Based on sodium dodecyl sulfate (SDS)-polyacrylamide electrophoretic properties, a widespread polymorphism (band 3 Memphis) has been previously described. It corresponds to a protein that has been hypothesized to be elongated in its N-terminal cytoplasmic domain. Band 3 from a heterozygote subject for this polymorphism and that displays a normal reactivity towards stilbene disulfonates has been isolated and its primary structure determined by protein chemistry. Reverse-phase high performance liquid chromatography tryptic peptide mapping showed, as the only difference with controls, that the enzymatic cleavage between the two N-terminal peptides did not occur, yielding a 69 residue-long fragment. Further cleavages of this peptide (cyanogen bromide, V8 protease), amino acid composition, and sequence analyses demonstrated that the lysine at position 56 was replaced by a glutamic acid. Thus, surprisingly, a single amino acid change is responsible for the large difference in the electrophoretic behavior. This result suggests that single amino acid substitutions may similarly be involved in the structural modification of several other protein variants, described as elongated or shortened based only on SDS-polyacrylamide electrophoresis studies. When deletions/insertions were confirmed by sequence analysis, their extent was often different from that expected from electrophoresis.
Effects of hematopoietic growth factors on the survival of primitive stem cells in liquid suspension culture
Authors: Bodine DM , Crosier PS , Clark SC
Published: October 2019

We have examined the effects of 10 different growth factors either alone or in combination on colony-forming unit-spleen (CFU-S) and repopulating stem cell survival in vitro. Either interleukin-3 (IL-3), granulocyte-colony-stimulating factor (G-CSF), or IL-4 alone support CFU-S in vitro. The effects of IL-3 or G-CSF could be neutralized by adding antibodies against IL-3 or G-CSF, respectively. However, the effects of IL-4 could be neutralized with antibodies to IL-4 as well as with antibodies to IL-3 and G-CSF. The combinations of IL-3 and IL-6, IL-3 and G-CSF, IL-3 and IL-1 alpha, IL-3 and granulocyte-macrophage CSF (GM-CSF), and IL-4 and IL-6 acted synergistically to increase CFU-S number. Addition of macrophage inflammatory protein-1 alpha (MIP-1 alpha) to IL-3 and IL-6 inhibited the increase in CFU-S number. Repopulating stem cell function was measured in a competitive repopulation assay. Either IL-3 or IL-4 alone could preserve stem cell function in vitro. The combinations of IL-3 and IL-6, and IL-3 and G- CSF increased stem cell function approximately twofold. The combinations of IL-3 + G-CSF + IL-6, and IL-4 and IL-6 (both of which increased CFU-S number fivefold to 10-fold) decreased stem cell function approximately fourfold. These results demonstrate that certain combinations of growth factors can increase CFU-S number at the expense of stem cell function.
Hematopoiesis in asymptomatic cats infected with feline immunodeficiency virus
Authors: Linenberger ML , Shelton GH , Persik MT , Abkowitz JL
Published: October 2019

Anemia and neutropenia often develop in cats that are infected with the feline immunodeficiency virus (FIV), a lentivirus biologically similar to the human immunodeficiency virus (HIV). To assess the role of FIV in the pathogenesis of these abnormalities, marrow culture studies were performed on nine asymptomatic, hematologically normal cats that were chronically infected with FIV. In these experiments, the frequencies of granulocyte/macrophage progenitors (CFU-GM) and early and late erythroid progenitors (CFU-E and BFU-E, respectively) were equivalent to progenitor frequencies in simultaneously studied uninfected control cats. Asymptomatic FIV infection was not associated with a change in the cell-cycle kinetics of CFU-E, BFU-E, or CFU-GM, nor was there an alteration in the dose-response of BFU-E or CFU-GM to hematopoietic growth factors present in fibroblast-derived conditioned medium. Sera from FIV-infected cats supported progenitor growth in vitro as well as normal cat sera. Furthermore, there was no evidence that these sera contained complement-fixing antibodies that recognized hematopoietic progenitors. Therefore, these data show that the in vitro behavior of hematopoietic progenitors is not affected by FIV infection alone, and they are in agreement with recent evidence that human progenitors are not a major target of HIV infection. It is likely that factors associated with progressive immunodeficiency, opportunistic infections, nutritional deficiencies, or malignancies play significant roles in the cytopenias that develop during the symptomatic disease induced by FIV, and by analogy, HIV. Prospective marrow culture studies of FIV-infected cats that develop hematologic abnormalities should provide a valuable animal model of acquired immunodeficiency syndrome-associated hematologic disorders.
The unique red cell heterogeneity of SC disease: crystal formation, dense reticulocytes, and unusual morphology
Authors: Lawrence C , Fabry ME , Nagel RL
Published: October 2019

Knowledge concerning SS (homozygous for the beta s gene) red blood cell (RBC) heterogeneity has been useful for understanding the pathophysiology of sickle cell anemia. No equivalent information exists for RBCs of the compound heterozygote for the beta s and beta c genes (SC) RBCs. These RBCs are known to be denser than most cells in normal blood and even most cells in SS blood (Fabry et al, J Clin Invest 70:1284, 1981). We have analyzed the characteristics of SC RBC heterogeneity and find that: (1) SC cells exhibit unusual morphologic features, particularly the tendency for membrane “folding” (multifolded, unifolded, and triangular shapes are all common); (2) SC RBCs containing crystals and some containing round hemoglobin (Hb) aggregates (billiard-ball cells) are detectable in circulating SC blood; (3) in contrast to normal reticulocytes, which are found mainly in a low-density RBC fraction, SC reticulocytes are found in the densest SC RBC fraction; and (4) both deoxygenation and replacement of extracellular Cl- by NO3- (both inhibitors of K:Cl cotransport) led to moderate depopulation of the dense fraction and a dramatic shift of the reticulocytes to lower density fractions. We conclude that the RBC heterogeneity of SC disease is very different from that of SS disease. The major contributions of properties introduced by HbC are “folded” RBCs, intracellular crystal formation in circulating SC cells, and apparently a very active K:Cl cotransporter that leads to unusually dense reticulocytes.
Migration of primed human eosinophils across cytokine-activated endothelial cell monolayers
Authors: Moser R , Fehr J , Olgiati L , Bruijnzeel PL
Published: October 2019

Eosinophils are known to adhere to cytokine-activated endothelium. Whereas transendothelial migration for neutrophils is an inevitable consequence of this endothelial-dependent adherence, this has not yet been shown for eosinophils. By means of human umbilical vein endothelial cells (HUVE) grown to confluence on microporous filters as an in vitro model of leukocytic migration across postcapillary venules, we have characterized the conditions leading to endothelium-driven transmigration of blood eosinophils from normals and from patients with allergic asthma. Freshly isolated eosinophils from nonallergic donors adhered to interleukin-1 (IL-1) and tumor necrosis factor-activated HUVE, but did not penetrate these monolayers. In contrast, eosinophils from allergic asthma patients showed an increased adherence and transmigration capacity. This increased functional competence was not caused by a difference in density phenotype, because the eosinophils from both groups showed a comparable density distribution over discontinuous Percoll gradients. Moreover, no difference existed within one group among eosinophils harvested from the Percoll density bands 1.080, 1.085, and 1.090 g/mL in terms of transendothelial migration. In vitro cultivation of freshly isolated eosinophils from nonallergic individuals in the presence of granulocyte-macrophage colony- stimulating factor (GM-CSF) and IL-3 induced a stepwise decrease of the density distribution over such gradients. In contrast, eosinophils from patients with allergic asthma directly shifted to a final density of 1.075 g/mL within 24 hours of culture. Notwithstanding the kinetics of density changes, eosinophils from nonallergic donors already expressed the capacity to transmigrate IL-1-activated HUVE monolayers 20 hours after cultivation with different combinations of GM-CSF, IL-3, and IL- 5. Inhibition studies with monoclonal antibodies showed that endothelium-driven transmigration of eosinophils predominantly implicates CD11/CD18 structures on the eosinophil surface, whereas no significant inhibition was found with the anti-VLA-4 monoclonal antibody HP2/1. From cytofluorometric studies, we conclude that spontaneous transmigration of eosinophils from allergic asthma patients is not accompanied by quantitative upregulation of these antigens. Taken together, these results allow the conclusion that blood eosinophils from allergic asthma patients have undergone in vivo priming, mimicked in vitro by cytokines such as GM-CSF, IL-3, and IL-5, leading to induction of the capacity to migrate across cytokine- activated HUVE monolayers.
Sustained human hematopoiesis in sheep transplanted in utero during early gestation with fractionated adult human bone marrow cells
Authors: Srour EF , Zanjani ED , Brandt JE , Leemhuis T , Briddell RA , Heerema NA , Hoffman R
Published: October 2019

Sheep were transplanted in utero during early gestation with subpopulations of adult human bone marrow (BM) cells enriched for human progenitor and hematopoietic stem cells (HSC). Chimerism was documented in three of seven transplanted fetuses using monoclonal antibodies against human-specific hematopoietic cell lineages and/or cytogenetic analysis of BM and peripheral blood cells of recipients. Only chimeric sheep BM cells expressing CD45 (6.0% of total BM cells) formed human hematopoietic colonies in response to human recombinant cytokines as determined by cytogenetic analysis. Sorted CD45+ BM cells developed human T-cell colonies containing CD3+, CD4+, and CD8+ cells. DNA from chimeric BM cells obtained 3 months after birth displayed a finger printing pattern identical to that of DNA from the human donor of the HSC graft. These studies indicate that first trimester sheep fetuses are tolerant of adult human HSC grafts, thus permitting the creation of xenogeneic chimera expressing human myeloid and lymphoid lineages. The present findings also suggest that HSC grafts from immunologically competent, HLA-mismatched adult donors may be useful for correcting human genetic diseases in utero during early gestation.
Absolute requirement of CD11/CD18 adhesion molecules, FcRII and the phosphatidylinositol-linked FcRIII for monoclonal antibody-mediated neutrophil antihuman tumor cytotoxicity
Authors: Kushner BH , Cheung NK
Published: October 2019

We have previously shown that 3F8, a murine IgG3, monoclonal antibody (MoAb) specific for the ganglioside GD2, mediates tumor cell kill in vitro and in vivo. We now describe receptor requirements of polymorphonuclear leukocytes (PMN) in 3F8-mediated cytotoxicity (ADCC) of human GD2 (+) melanoma and neuroblastoma cell lines. PMN from a child with leukocyte adhesion deficiency (LAD) were devoid of CD11/CD18 adhesion molecules and mounted no detectable ADCC. MoAb to CD11b, CD11c, and CD18 each efficiently blocked ADCC by normal PMN. In contrast, a panel of different MoAbs to CD11a had no significant inhibitory effect on ADCC, a finding consistent with the low-to-absent expression of the CD11a ligand, intercellular adhesion molecule-1, on the target cells. Granulocyte-macrophage colony-stimulating factor (GM- CSF) significantly increased the expression of CD11b, CD11c, and CD18 on normal PMN, decreased the expression of Fc receptors (FcR), and enhanced ADCC by normal but not by LAD PMN. MoAbs to FcRII and FcRIII each efficiently blocked ADCC; anti-FcRI MoAb had no effect. Flow cytometry using anti-FcRII MoAb versus anti-FcRIII MoAb did not show cross competition, suggesting that inhibition of ADCC was not a steric effect resulting from FcRII proximity to FcRIII. PMN deficient in FcRIII (obtained from patients with paroxysmal nocturnal hemoglobinuria) and PMN depleted of FcRIII by treatment with elastase or phosphatidylinositol (PI)-specific phospholipase C produced low ADCC, supporting a role for the PI-liked FcRIII. Thus, optimal ADCC using human PMN, human solid tumor cells, and a clinically active MoAb (conditions that contrast with the heterologous antibodies and nonhuman or nonneoplastic targets used in most models of PMN ADCC) required CD11b, CD11c, FcRII, and the PI-linked FcRIII. Furthermore, in this clinically relevant system, GM-CSF enhancement of antitumor PMN ADCC correlated with increased expression of CD11/CD18 molecules.
Posttranslational processing of defensins in immature human myeloid cells
Authors: Valore EV , Ganz T
Published: October 2019

Human neutrophil promyelocytes synthesize, process, and package several microbicidal proteins, including the highly abundant defensins, into their azurophil granules. As deduced from their cDNA sequences, defensins are initially synthesized as 94 amino acid (aa) precursors that must undergo extensive processing. We performed metabolic labeling studies of defensin synthesis in the human promyelocytic cell line HL- 60 and in chronic myeloid leukemia cells, and showed that preprodefensins are processed to mature 29 to 30 aa defensins over 4 to 24 hours via two major intermediates: a 75 aa prodefensin generated by the cleavage of the signal sequence, and a 56 aa prodefensin that results from a subsequent preaspartate proteolytic cleavage. Almost all of the 75 aa form was found in the cytoplasmic/microsomal fraction, whereas the 56 aa prodefensin and mature defensins predominated in the granule-enriched fraction. The 75 aa prodefensin was also selectively released into the culture supernatant. Treatment of HL-60 cells with monensin, chloroquine, or ammonium chloride, substances that neutralize acidic subcellular compartments, partially blocked conversion of the 75 aa prodefensin into 56 aa prodefensin, but did not increase the extracellular release of the 75 aa form. Further studies will be required to determine the role of this processing pathway in subcellular targeting to azurophil granules and avoidance of autocytotoxicity.
Antisense oligodeoxynucleotide combination therapy of primary chronic myelogenous leukemia blast crisis in SCID mice
Authors: Skorski T , Nieborowska-Skorska M , Wlodarski P , Zon G , Iozzo RV , Calabretta B
Published: October 2019

The proliferation of chronic myelogenous leukemia (CML) cells and the transformation of normal hematopoietic cells by BCR-ABL appear to require the expression of a functional MYC protein, suggesting an approach to treatment of Philadelphia leukemias based on simultaneous targeting of BCR-ABL and c-MYC. To test this hypothesis, CML-blast crisis (CML-BC) primary cells were treated in vitro with bcr-abl and c- myc antisense phosphorothioate oligodeoxynucleotides ([S]ODNs), individually or in combination. Compared with antisense ODNs targeting of individual oncogenes, downregulation of both BCR-ABL and c-MYC by specific antisense [S]ODNs resulted in a synergistic antiproliferative effect. Colony formation of normal bone marrow cells was not affected by either treatment. To assess the therapeutic potential of multiple oncogene downregulation, SCID mice injected with CML-BC primary cells were treated systematically with equal doses of bcr-abl or c-myc antisense [S]ODNs or with a combination of both antisense [S]ODNs. Compared with mice treated with individual compounds, the disease process was significantly retarded in the group treated with both [S]ODNs as revealed by flow cytometry, clonogenic assay, and RT-PCR analysis to detect leukemic cells in mouse tissue cell suspensions. These effects correlated with a markedly increased survival of leukemic mice treated with both antisense [S]ODNs. Leukemic cells harvested from antisense [S]ODN-treated mice were sensitive to the effects of antisense [S]ODNs in vitro, suggesting that the treatment can be successfully repeated. These data demonstrate the therapeutic potential of targeting multiple cooperating oncogenes.
Essential Role of the Thymus to Reconstitute Naive (CD45RA+) T-Helper Cells After Human Allogeneic Bone Marrow Transplantation
Authors: Heitger Andreas , Neu Nikolaus , Kern Hannelore , Panzer-Grümayer Eva-Renate , Greinix Hildegard , Nachbaur David , Niederwieser Dietger , Fink Franz Martin
Published: October 2019

To contribute to the understanding of the role of the thymus in humans in the reconstitution of naive (CD45RA+) T cells after bone marrow transplantation (BMT), we compared T-cell regeneration in a unique situation, namely a thymectomized cancer patient (15 years old), with that of thymus-bearing patients after allogeneic BMT. These cases shared features of transplantation (total body irradiation, HLA-matched donors, and graft-versus-host disease prophylaxis with cyclosporine A) and all had an uncomplicated posttransplantation course. As shown by fluorescence-activated cell sorting analyses, the thymectomized host failed to reconstitute CD45RA+ T-helper cells even 24 months after BMT (11% CD45RA+ of CD4+ cells). In this patient, preferentially CD45RO+ cells contributed to the recovery of CD4+ cells (206 of 261/μL at 6 months and 463 of 558/μL at 24 months after BMT, CD45RA+ of CD4+ cells), whereas CD45RA+ cells remained low (<60/μL). In contrast, nine thymus-bearing hosts (5 children and 4 adults) examined between 6 and 24 months after BMT effectively reconstituted CD4+/CD45RA+ cells according to their normal age-related range (≥28% in adults and ≥50% in children). Five of these were analyzed sequentially at 6 and 9 months after BMT. Within this period, CD45RA+ cells increasingly contributed to the recovery of CD4+ cells (median, +21%), even when total CD4+ cells decreased. With respect to T-cytotoxic/suppressor cells, the thymectomized host retained the capacity to recover CD45RA+ cells (137 of 333/μL at 6 months and 596 of 1,046/μL at 24 months after BMT, CD45RA+ of CD8+ cells), a proportion similar to that seen in thymus-bearing hosts. These findings suggest that a thymus-independent pathway exists to regenerate CD45RA+ T-cytotoxic/suppressor cells, but residual thymus is essential to reconstitute naive (CD45RA+) T-helper cells after BMT in humans.
FcγRIIIa-158V/F Polymorphism Influences the Binding of IgG by Natural Killer Cell FcγRIIIa, Independently of the FcγRIIIa-48L/R/H Phenotype
Authors: Koene Harry R. , Kleijer Marion , Algra Johan , Roos Dirk , E.G.Kr. von dem Borne Albert , de Haas Masja
Published: October 2019

We analyzed a genetic polymorphism of Fcγ receptor IIIa (CD16) that is present on position 158 (Phe or Val) in the membrane-proximal, IgG-binding domain. With a polymerase chain reaction–based allele-specific restriction analysis assay we genotyped 87 donors and found gene frequencies of 0.57 and 0.43 for FcγRIIIA-158F and −158V, respectively. A clear linkage was observed between the FcγRIIIA-158F and −48L genotypes on the one hand and the FcγRIIIA-158V and −48H or −48R genotypes on the other hand (χ2 test; P < .001). To determine the functional consequences of this FcγRIIIa-158V/F polymorphism, we performed IgG binding experiments with natural killer (NK) cells from genotyped donors. All donors were also typed for the recently described triallelic FcγRIIIa-48L/R/H polymorphism. NK cells were treated with lactic acid to remove cell-associated IgG. FcγRIIIaNK158F bound significantly less IgG1, IgG3, and IgG4 than did FcγRIIIaNK-158V, irrespective of the FcγRIIIa-48 phenotype. Moreover, freshly isolated NK cells from FcγRIIIa-158VV individuals carried significantly more cytophilic IgG than did NK cells from FcγRIIIa-158FF individuals. In addition, CD16 monoclonal antibody (MoAb) MEM154 bound more strongly to FcγRIIIa-158V, compared with -158F, again independently of the FcγRIIIa-48 phenotype. The binding of MoAb B73.1 was not influenced by the FcγRIIIa-158V/F polymorphism, but proved to depend solely on the amino acid present at position 48 of FcγRIIIa. In conclusion, the previously reported differences in IgG binding among the three FcγRIIIa-48L/R/H isoforms are a consequence of the linked, biallelic FcγRIIIa-158V/F polymorphism at amino-acid position 158.
Upregulation of the Elongation Factor-1α Gene by p53 in Association With Death of an Erythroleukemic Cell Line
Authors: Kato Mitsuo V. , Sato Hiromi , Nagayoshi Mariko , Ikawa Yoji
Published: October 2019

Genes upregulated by p53 were screened using an erythroleukemic cell line (1-2-3) that expresses only the temperature-sensitive p53 by the mRNA differential display method. One of the upregulated genes was identified as the elongation factor-1α (EF-1α) gene, an essential component of the eukaryotic translation apparatus. Three p53-responsive elements were found in the mouse EF-1α gene and in the corresponding human, rat, and frog genes. These elements conferred the capacity for induction by p53. EF-1α is also a microtubule-severing protein. Upon the temperature-shift, the cells developed the morphology and the localization of α-tubulin similar to those of the cells treated with vincristine, a drug that affects microtubules. The microtubule-severing associated with upregulation of EF-1α by p53 may be a cause of the cell death.
Aberrations of the B-Cell Receptor B29 (CD79b) Gene in Chronic Lymphocytic Leukemia
Authors: Thompson Alexis A. , Talley Jeaniene A. , Do Ha Nancy , Kagan H. Lee , Kunkel Lori , Berenson James , Cooper Max D. , Saxon Andrew , Wall Randolph
Published: October 2019

Leukemic B cells in chronic lymphocytic leukemia (B-CLL) typically exhibit low or undetectable surface Ig. Because the B29 (CD79b and Igβ) and mb-1 (CD79a and Igα) gene products are required for surface Ig display in the B-cell receptor complex (BCR), we analyzed the expression of these genes in B-CLL cells. The majority (83%) of the randomly selected B-CLL patient samples analyzed exhibited low or undetectable surface BCR measured by μ heavy chain and B29 expression. Levels of mb-1 mRNA in these B-CLL samples with low surface BCR were similar to those in normal B cells. Among those with decreased surface expression, B29 mRNA was not detected in half of these B-CLL samples. The remaining B-CLL samples with diminished surface BCR contained normal levels of B29 mRNA. Further analysis of cDNA clones from the majority of these latter samples contained point mutations, insertions, or deletions that were largely located in the B29 transmembrane and cytoplasmic domains. These results indicate the occurrence of somatic mutations predicted to affect B29 expression and/or function in the majority of B-CLL and suggest that these aberrations underlie the diminished surface BCR display and loss of BCR signaling characteristic of this leukemia.
Hypermethylation of the p15INK4B Gene in Myelodysplastic Syndromes
Authors: Uchida Toshiki , Kinoshita Tomohiro , Nagai Hirokazu , Nakahara Yohsuke , Saito Hidehiko , Hotta Tomomitsu , Murate Takashi
Published: October 2019

Previous studies have shown that the cyclin-dependent kinase inhibitor (CDKI) genes p15INK4B and p16INK4A are frequently inactivated by genetic alterations in many malignant tumors and that they are candidate tumor-suppressor genes. Although genetic alterations in these genes may be limited to lymphoid malignancies, it has been reported that their inactivation by aberrant methylation of 5′ CpG islands may be involved in various hematologic malignancies. In this study, we investigated the p15INK4B and p16INK4A genes to clarify their roles in the pathogenesis of myelodysplastic syndrome (MDS). Southern blotting analysis showed no gross genetic alterations in either of these genes. However, hypermethylation of the 5′ CpG island of the p15INK4B gene occurred frequently in patients with MDS (16/32 [50%]). Interestingly, the p15INK4B gene was frequently methylated in patients with high-risk MDS (refractory anemia with excess blasts [RAEB], RAEB in transformation [RAEB-t], and overt leukemia evolved from MDS; 14/18 [78%]) compared with patients with low-risk MDS (refractory anemia [RA] and refractory anemia with ring sideroblast [RARS]; 1/12 [8%]). Furthermore, methylation status of the p15INK4B gene was progressed with the development of MDS in most patients examined. In contrast, none of the MDS patients showed apparent hypermethylation of the p16INK4A gene. These results suggest that hypermethylation of the p15INK4B gene is involved in the pathogenesis of MDS and is one of the important late events during the development of MDS.
Marrow Transplants From Unrelated Donors for Treatment of Philadelphia Chromosome-Positive Acute Lymphoblastic Leukemia
Authors: Sierra Jorge , Radich Jerry , Hansen John A. , Martin Paul J. , Petersdorf Effie W. , Bjerke Jeanie , Bryant Eileen , Nash Richard A. , Sanders Jean E. , Storb Rainer , Sullivan Keith M. , Appelbaum Frederick R. , Anasetti Claudio
Published: October 2019

Transplantation of marrow from unrelated donors was investigated in patients with Philadelphia chromosome-positive (Ph1+) acute lymphoblastic leukemia (ALL) who lacked a suitable family donor. Eighteen patients underwent transplantation at our center between 1988 and 1995. The median patient age was 25 years (range, 1.7 to 51 years). Seven patients were in first complete remission, 1 in second remission, 3 in first relapse, and the remaining 7 had more advanced or chemotherapy refractory leukemia at transplant. All patients were conditioned with cyclophosphamide and total body irradiation followed by marrow transplants from closely HLA-matched, unrelated volunteers. Posttransplant graft-versus-host disease (GVHD) prophylaxis included methotrexate with either cyclosporine or FK506. Graft failure was not observed. Severe (grades III-IV) GVHD appeared in 6 of 17 evaluable patients and chronic extensive GVHD in 7 of 13 patients at risk. Five patients had recurrent ALL after transplantation and another 4 died from causes other than leukemia. Six patients transplanted in first remission, 2 in first relapse, and 1 in second remission remain alive and leukemia-free at a median follow-up of 17 months (range, 9 to 73 months). The probability of leukemia-free survival at 2 years is 49% ± 12%. These data indicate that unrelated donor marrow transplantation is an effective treatment option for patients with early stage Ph1+ ALL without a family match and suggest that in such patients an unrelated donor search should be initiated as soon as possible after diagnosis.
Novel Interaction of Apolipoprotein(a) With β-2 Glycoprotein I Mediated by the Kringle IV Domain
Authors: Köchl Silvano , Fresser Friedrich , Lobentanz Eva , Baier Gottfried , Utermann Gerd
Published: October 2019

Lipoprotein(a) [Lp(a)], which has been shown to interact with fibrin(ogen) and other components of the blood clotting cascade, is a major independent risk factor for atherothrombotic disease in humans. The physiological function(s) of Lp(a), as well as the precise mechanism(s) by which high plasma levels of Lp(a) increase risk are unknown. Identification of further potential apo(a)-protein ligands may be crucial to illuminate apo(a)'s function(s) and pathophysiological properties. We used the repetitive apo(a) kringle IV type 2, which is variable in number in apo(a), to screen a human liver cDNA library by the yeast two-hybrid interaction trap system. Among 11 positive clones that emerged from the screen, eight clones were identified as β-2 glycoprotein I and one as fibronectin. Coimmunoprecipitation experiments confirmed that β-2 glycoprotein I and apo(a)/Lp(a) interact in human plasma and in cell culture supernatants of COS-1 cells, which ectopically expressed apo(a). The apo(a)-β2-glycoprotein I interaction indicates new potential roles for Lp(a) in fibrinolysis and autoimmunity.
Interleukin-6 Downregulates Factor XII Production by Human Hepatoma Cell Line (HepG2)
Authors: Citarella Franca , Felici Angelina , Brouwer Mieke , Wagstaff John , Fantoni Antonio , Hack C. Erik
Published: October 2019

Involvement of the contact system of coagulation in the pathogenesis of various inflammatory diseases is suggested by reduced plasma levels of factor XII (Hageman factor) and prekallikrein generally considered to result from activation of the contact system. However, in many of these diseases patients develop an acute-phase response and, therefore, an alternative explanation for the decreased levels of factor XII could be the downregulation of factor XII gene expression in the liver as described for negative acute-phase proteins. We report here that interleukin-6 (IL-6), the principal cytokine mediating the synthesis of most acute-phase proteins in the liver, downregulates the production of factor XII by the human hepatoma cell line HepG2 by up to 75%. The decrease in protein secretion correlated with an equivalent decrease of factor XII mRNA likely indicating a pretranslational control of factor XII gene expression by IL-6. Downregulation of factor XII production by IL-6 in vitro parallelled that of transthyretin, a known negative acute-phase protein. Moreover, we show that, in patients developing an acute-phase response after immunotherapy with IL-2, plasma levels of factor XII correlate (r = .76, P < .0001) with those of transthyretin. Taken together, these results suggest that factor XII behaves as a negative acute-phase protein.
A Role for T-Helper Type-1 and Type-2 Cytokines in the Regulation of Human Monocyte Apoptosis
Authors: Estaquier Jérôme , Ameisen Jean Claude
Published: October 2019

T-helper type-1 (Th1) and type-2 (Th2) cytokines, respectively, favor T-cell–mediated immunity and defense against intracellular pathogens or antibody-mediated immunity and defense against extracellular pathogens. Here we report that type-1 and type-2 cytokines also exert a regulatory effect on human monocyte survival. Interleukin-12 (IL-12) enhanced survival in long-term (10 days) cultures of adherent monocytes, whereas IL-10 induced death by apoptosis. In short-term cultures (2 days), the Th2 cytokines, IL-10 and IL-4, enhanced apoptosis; however, the Th1 cytokines, IL-12 and IL-2 only showed a reducing effect on monocyte apoptosis in culture conditions that decreased monocyte adhesion leading to increased levels of spontaneous apoptosis; finally, the Th1 cytokine, interferon-γ (IFN-γ), acted in a dose-dependent fashion: At high concentrations, IFN-γ enhanced apoptosis, which is an effect related to IL-10 secretion and reduced by antibodies to IL-10. Th1 cytokines reduced monocyte apoptosis induced by several stimuli: IL-2 reduced apoptosis induced by either IL-10 or high concentrations of IFN-γ, IL-12 reduced apoptosis induced by either the ligation of the Fas (CD95) molecule or γ-irradiation, and IFN-γ (at low doses that did not trigger apoptosis) reduced apoptosis induced by γ-irradiation. These findings suggest that the regulatory role of type-1 and type-2 cytokines on the development of immune responses and inflammatory reactions also involves the regulation of monocyte death by apoptosis.
Immunohistochemical Detection of the Epstein-Barr Virus–Encoded Latent Membrane Protein 2A in Hodgkin's Disease and Infectious Mononucleosis
Authors: Niedobitek G. , Kremmer E. , Herbst H. , Whitehead L. , Dawson C.W. , Niedobitek E. , von Ostau C. , Rooney N. , Grässer F.A. , Young L.S.
Published: October 2019

We describe two new monoclonal antibodies specific for the Epstein-Barr virus (EBV)-encoded latent membrane protein 2A (LMP2A) that are suitable for the immunohistochemical analysis of routinely processed paraffin sections. These antibodies were applied to the immunohistochemical detection of LMP2A in Hodgkin's disease (HD). LMP2A-specific membrane staining was seen in the Hodgkin and Reed-Sternberg (HRS) cells of 22 of 42 (52%) EBV-positive HD cases, but not in 39 EBV-negative HD cases. In lymphoid tissues from patients with acute infectious mononucleosis (IM), interfollicular immunoblasts were shown to express LMP2A. This is the first demonstration of LMP2A protein expression at the single-cell level in EBV-associated lymphoproliferations in vivo. The detection of LMP2A protein expression in HD and IM is of importance in view of the proposed role of this protein for maintaining latent EBV infection and its possible contribution for EBV-associated transformation. Because LMP2A provides target epitopes for EBV-specific cytotoxic T cells, the expression of this protein in HRS cells has implications for the immunotherapeutic approaches to the treatment of HD.
CD44 Regulates Hematopoietic Progenitor Distribution, Granuloma Formation, and Tumorigenicity
Authors: Schmits Rudolf , Filmus Jorge , Gerwin Nicole , Senaldi Giorgio , Kiefer Friedemann , Kundig Thomas , Wakeham Andrew , Shahinian Arda , Catzavelos Charles , Rak Janusz , Furlonger Caren , Zakarian Arsen , Simard John J.L. , Ohashi Pamela S. , Paige Christopher J. , Gutierrez-Ramos Jose C. , Mak Tak W.
Published: October 2019

CD44 is expressed in various isoforms on numerous cell types and tissues during embryogenesis and in the mature organism. CD44 may also be involved in tumor growth. To study the multiple roles of CD44, we abolished expression of all known isoforms of CD44 in mice by targeting exons encoding the invariant N-terminus region of the molecule. Surprisingly, mice were born in Mendelian ratio without any obvious developmental or neurological deficits. Hematological impairment was evidenced by altered tissue distribution of myeloid progenitors with increased levels of colony-forming unit–granulocyte-macrophage (CFU-GM) in bone marrow and reduced numbers of CFU-GM in spleen. Fetal liver colony-forming unit–spleen and granulocyte colony-stimulating factor mobilization assays, together with reduced CFU-GM in peripheral blood, suggested that progenitor egress from bone marrow was defective. In what was either a compensatory response to CD44 deficiency or an immunoregulatory defect, mice also developed exaggerated granuloma responses to Cryotosporidium parvum infection. Finally, tumor studies showed that SV40-transformed CD44-deficient fibroblasts were highly tumorigenic in nude mice, whereas reintroduction of CD44s expression into these fibroblasts resulted in a dramatic inhibition of tumor growth.
In Vitro Senescence and Apoptotic Cell Death of Human Megakaryocytes
Authors: Zauli Giorgio , Vitale Marco , Falcieri Elisabetta , Gibellini Davide , Bassini Alessandra , Celeghini Claudio , Columbaro Marta , Capitani Silvano
Published: October 2019

To investigate the fate of human megakaryocytes, CD34+ hematopoietic progenitor cells were purified from the peripheral blood or bone marrow of healthy donors and seeded in serum-free chemically defined suspension cultures. In the presence of thrombopoietin (TPO; 100 ng/mL), CD34-derived cells showed an eightfold numerical expansion and a progressive maturation along the megakaryocytic lineage. Megakaryocyte maturation was characterized ultrastructurally by the presence of a demarcation membrane system and phenotypically by a high surface expression of αIIbβ3 integrin. The number of mature megakaryocytes peaked at days 12 to 15 of culture. On the other hand, the number of platelets released in the culture supernatant by CD34-derived megakaryocytes peaked at days 18 to 21, when a high percentage of megakaryocytes showed the characteristic features of apoptosis, as evaluated by electron microscopy, terminal deoxynucleotidyl transferase (TdT)-mediated d-UTP-biotin nick end-labeling technique (TUNEL) and uptake of propidium iodide. In other experiments, primary αIIbβ3+ megakaryocytic cells were directly purified from the bone marrow aspirates of normal donors and seeded in serum-free suspension cultures. In the absence of cytokines, αIIbβ3+ megakaryocytes progressively underwent apoptotic cell death. The addition of TPO but not interleukin-3 or erythropoietin showed some protection of αIIbβ3+ cells from apoptosis at early culture times (days 2 to 4), but it did not show any significant effect at later time points. These findings suggest that the terminal phase of the megakaryocyte life span is characterized by the onset of apoptosis, which can be modulated only to a certain extent by TPO.
Characterization of Signal Transduction Pathways in Human Bone Marrow Endothelial Cells
Authors: Liu Zhong-Ying , Ganju Ramesh K. , Wang Jian-Feng , Schweitzer Karin , Weksler Babette , Avraham Shalom , Groopman Jerome E.
Published: October 2019

Human bone marrow endothelial cells immortalized with the T antigen of SV40 (TrHBMEC) have previously been characterized by us with regard to their properties that are similar to primary marrow endothelial cells and their utility as a model system. We now report that TrHBMEC express a recently discovered signal transduction molecule termed RAFTK (related adhesion focal tyrosine kinase), also called Pyk2 or CAK-β. RAFTK, the second member of the focal adhesion kinase (FAK) family, is known to be activated in response to calcium flux in neuronal cells and integrin stimulation in megakaryocytes and B cells. We have studied the effects of cytokines on RAFTK activation in TrHBMEC. Treatment of TrHBMEC with the vascular endothelial growth factor (VEGF ), as well as the VEGF-related protein (VRP), the recently identified ligand for the FLT-4 receptor, resulted in enhanced tyrosine phosphorylation of RAFTK. Similar changes in RAFTK phosphorylation were observed upon stimulation of TrHBMEC with basic fibroblast growth factor (bFGF ) or oncostatin M (OSM). Stimulation of these cells with growth factors also resulted in an increase in RAFTK activity and the c-Jun NH2 -terminal kinase (JNK). RAFTK coimmunoprecipitated with the cytoskeletal protein paxillin through its C-terminal proline-rich domain in TrHBMEC. These results suggest that, in marrow endothelium, activation of RAFTK by VEGF, VRP, OSM, and bFGF represents a new element in the signal transduction pathways used by these growth factors and likely acts to coordinate signaling from their surface receptors to the cytoskeleton, thereby modulating cell growth and function.
Increased Recruitment of Hematopoietic Progenitor Cells Underlies the Ex Vivo Expansion Potential of FLT3 Ligand
Authors: Haylock David N. , Horsfall Martyn J. , Dowse Tracey L. , Ramshaw Hayley S. , Niutta Silvana , Protopsaltis Sandra , Peng Li , Burrell Christopher , Rappold Irene , Buhring Hans-Jorg , Simmons Paul J.
Published: October 2019

The ligand for flt-3 (FLT3L) exhibits striking structural homology with stem cell factor (SCF ) and monocyte colony-stimulating factor (M-CSF ) and also acts in synergy with a range of other hematopoietic growth factors (HGF ). In this study, we show that FLT3L responsive hematopoietic progenitor cells (HPC) are CD34+CD38−, rhodamine 123dull, and hydroperoxycyclophosphamide (4-HC) resistant. To investigate the basis for the capacity of FLT3L to augment the de novo generation of myeloid progenitors from CD34+CD38− cells, single bone marrow CD34+CD38− cells were sorted into Terasaki wells containing serum-free medium supplemented with interleukin-3 (IL-3), IL-6, granulocyte colony-stimulating factor (G-CSF ), SCF (4 HGF ) ± FLT3L. Under these conditions, FLT3L recruited approximately twofold more CD34+CD38− cells into division than 4 HGF alone. The enhanced proliferative response to FLT3L was evident by day 3 and was maintained at all subsequent time points examined. In accord with these findings, we also show that transduction of CD34+CD38− cells with the LAPSN retrovirus is enhanced by FLT3L. The results of these experiments therefore indicate that increased recruitment of primitive HPC into cell cycle underlies the ex vivo expansion potential of FLT3L and also its ability to improve retroviral transduction of HPC.
CD34 Expression Patterns During Early Mouse Development Are Related to Modes of Blood Vessel Formation and Reveal Additional Sites of Hematopoiesis
Authors: Wood Heather B. , May Gillian , Healy Lyn , Enver Tariq , Morriss-Kay Gillian M.
Published: October 2019

CD34 is a cell surface glycoprotein that is selectively expressed within the human hematopoietic system on stem and progenitor cells, and in early blood vessels. To elucidate its functions during early blood vessel formation and hematopoiesis, we analyzed the expression patterns, in day 8 to day 10 mouse embryos, of CD34 RNA by in situ hybridization and protein by immunohistochemistry using the monclonal antibody RAM 34. Levels of expression in embryonic blood vessels were correlated with the mode of vessel formation, being high in pre-endothelial cells and in vessels forming by vasculogenesis (particularly the dorsal aortae) or angiogenesis, but low in vessels forming by coalescence (the cardinal veins). CD34+ erythroid cells, presumably of yolk sac origin, were present in the liver of day 10 embryos; at the same stage, putative definitive hematopoietic cells, strongly CD34+, were present in the para-aortic mesenchyme. Possible sites of hemangioblastic differentiation were detected in the form of CD34+ endothelium-attached hematopoietic cells in the dorsal aorta and in two previously unreported locations, the proximal umbilical and vitelline arteries. These observations suggest functions for CD34 in relation to specific modes of blood vessel formation, and a hemangioblastic role in both embryonic and extraembryonic sites.
Soluble Monomeric P-Selectin Containing Only the Lectin and Epidermal Growth Factor Domains Binds to P-Selectin Glycoprotein Ligand-1 on Leukocytes
Authors: Mehta Padmaja , Patel Kamala D. , Laue Thomas M. , Erickson Harold P. , McEver Rodger P.
Published: October 2019

Under shear stress, leukocytes use P-selectin glycoprotein ligand-1 (PSGL-1) to tether to and roll on P-selectin expressed on activated platelets or endothelial cells. P-selectin has an NH2-terminal lectin domain, an epidermal growth factor (EGF)-like motif, nine consensus repeats (CRs), a transmembrane domain, and a cytoplasmic tail. To determine whether the CRs are required for P-selectin to bind PSGL-1, we expressed a soluble protein (Lec-EGF) that contained only the lectin and EGF domains, plus a short C-terminal epitope tag. Electron microscopy and hydrodynamic analysis confirmed that Lec-EGF was monomeric, as previously shown for soluble P-selectin (sPS) that contained the lectin and EGF domains plus all nine CRs. Fluid-phase Lec-EGF or sPS inhibited binding of oligomeric125I-labeled membrane-derived P-selectin (mPS) to PSGL-1 on neutrophils and binding of 125I-PSGL-1 to immobilized mPS. The IC50 for inhibiting binding of mPS to neutrophils was fivefold greater for Lec-EGF than for sPS, whereas the IC50 for inhibiting binding of mPS to purified PSGL-1 was indistinguishable for Lec-EGF and sPS. Under static or shear conditions, neutrophils used PSGL-1 to tether to or roll on Lec-EGF that was captured by an immobilized monoclonal antibody to the C-terminal epitope. These data show that P-selectin requires only the lectin and EGF domains to bind to PSGL-1.
Immunohistochemical Analysis of Interleukin-1β–Converting Enzyme/Ced-3 Family Protease, CPP32/Yama/Caspase-3, in Hodgkin's Disease
Authors: Chhanabhai Mukesh , Krajewski Stanislaw , Krajewska Maryla , Wang Hong-Gang , Reed John C. , Gascoyne Randy D.
Published: October 2019

The Caenorhabditis elegans cell death gene, Ced-3, encodes a protein homologous to mammalian interleukin-1β–converting enzyme (ICE), a cysteine protease implicated in programmed cell death (PCD). CPP32, also known as Yama, apopain, and Caspase-3, is a member of this family, has substrate specificities similar to Ced-3, and has been shown to have an active role in PCD. Evidence suggests that these proteases act downstream of inhibitors of PCD such as Bcl-2 and Bcl-xL , which are frequently expressed in Reed-Sternberg (RS) cells of Hodgkin's disease (HD). To date there have been no studies examining the role of the ICE/Ced-3 family of proteins, in particular CPP32, in HD. We examined 24 cases of HD with a classical immunophenotype and 6 cases of nodular lymphocyte predominant HD (NLPHD) for the expression of CPP32 in the RS cells and lymphohistiocytic (L&H) cells as detected by immunohistochemistry. Twenty two of 24 cases (92%) of HD expressed the protein in the RS cells, whereas the L&H cells in all 6 cases of NLPHD lacked expression of CPP32. These results provide further evidence that NLPHD is a phenotypically different disease distinct from classical forms of HD. The differential expression of the cell death protein CPP32 may be an important factor contributing to the apparently different clinical behaviour of NLPHD in contrast to classical HD. The lack of expression of CPP32 in NLPHD shares similarities with low-grade B-cell non-Hodgkin's lymphomas and may explain their common clinical course. Further studies are required to elucidate the significance of CPP32 in HD.
A Markedly Disrupted Skeletal Network With Abnormally Distributed Intramembrane Particles in Complete Protein 4.1-Deficient Red Blood Cells (Allele 4.1 Madrid): Implications Regarding a Critical Role of Protein 4.1 in Maintenance of the Integrity of the Red Blood Cell Membrane
Authors: Yawata Ayumi , Kanzaki Akio , Gilsanz Florinda , Delaunay Jean , Yawata Yoshihito
Published: October 2019

Electron microscopic (EM) studies were performed to clarify the interactions of membrane proteins in the red blood cell membrane structure in situ of a homozygous patient with total deficiency of protein 4.1 who carried a point mutation of the downstream translation initiation codon (AUG → AGG) of the protein 4.1 gene [the 4.1 (−) Madrid; Dalla Venezia et al, J Clin Invest 90:1713, 1992]. Immunologically, as expected, protein 4.1 was completely missing in the red blood cell membrane structure in situ. A markedly disrupted skeletal network was observed by EM using the quick-freeze deep-etching method and the surface replica method, although the number of spectrin molecules was only minimally reduced (395 ± 63/μm2; normal, 504 ± 36/μm2). The number of basic units in the skeletal network was strikingly reduced (131 ± 21/μm2; normal, 548 ± 39/μm2), with decreased small-sized units (17 ± 4/μm2; normal, 384 ± 52/μm2) and increased large-sized units (64% ± 14%; normal, 5% ± 1%). Concomitantly, immuno-EM disclosed striking clustering of spectrin molecules with aggregated ankyrin molecules in the red blood cell membrane structure in situ. Although no quantitative abnormalities in the number and size distribution of the intramembrane particles were observed, there was a disappearance of regular distribution, with many clusters of various sizes, probably reflecting the distorted skeletal network. Therefore, protein 4.1 suggests by EM to play a crucial role in maintenance of the normal integrity of the membrane structure in situ not only of the skeletal network but also of the integral proteins.