bluuebunny/crossref_metadata_2025 huggingface dataset:
Primitive Long-Term Culture Initiating Cells (LTC-ICs) in Granulocyte Colony-Stimulating Factor Mobilized Peripheral Blood Progenitor Cells Have Similar Potential for Ex Vivo Expansion as Primitive LTC-ICs in Steady State Bone Marrow
Authors: Prosper Felipe , Vanoverbeke Kirk , Stroncek David , Verfaillie Catherine M.
Published: October 2019

We have recently shown that more than 90% of long-term culture initiating cells (LTC-IC) mobilized in the peripheral blood (PB) of normal individuals express HLA-DR and CD38 antigens and can sustain hematopoiesis for only 5 weeks. However, 10% of LTC-IC in mobilized PB are CD34+HLA-DR− and CD34+CD38− and can sustain hematopoiesis for at least 8 weeks. We now examine the ex vivo expansion potential of CD34+HLA-DR+ cells (rich in mature LTC-IC) and CD34+HLA-DR− cells (rich in primitive LTC-IC) in granulocyte colony-stimulating factor (G-CSF ) mobilized PB progenitor cells (PBPC). Cells were cultured in contact with M2-10B4 cells (contact) or in transwells above M2-10B4 (noncontact) without and with interleukin-3 (IL-3) and macrophage inflammatory protein (MIP-1α) for 2 and 5 weeks. Progeny were evaluated for the presence of colony-forming cells (CFC) and LTC-IC. When CD34+HLA-DR+ PB cells were cultured in contact cultures without cytokines, a threefold expansion of CFC was seen at 2 weeks, but an 80% decrease in CFC was seen at week 5. Further, the recovery of LTC-IC at week 2 was only 17% and 1% at week 5. This confirms our previous observation that although CD34+HLA-DR+ mobilized PB cells can initiate long-term cultures, they are relatively mature and cannot sustain long-term hematopoiesis. In contrast, when CD34+HLA-DR− mobilized PB cells were cultured in contact cultures without cytokines, CFC expansion persisted until week 5 and 49% and 11% of LTC-IC were recovered at week 2 and 5, respectively. As we have shown for steady state bone marrow (BM) progenitors, recovery of LTC-IC was threefold higher when CD34+HLA-DR− PBPC were cultured in noncontact rather than contact cultures, and improved further when IL-3 and MIP-1α were added to noncontact cultures (96 ± 2% maintained at week 5). We conclude that although G-CSF mobilizes a large population of “mature” CD34+HLA-DR+ LTC-IC with a limited proliferative capacity, primitive CD34+HLA-DR− LTC-IC present in mobilized PB have similar characteristics as LTC-IC from steady state BM: (1) they can be maintained in noncontact cultures containing IL-3 and MIP-1α for at least 5 weeks; (2) they are subject to the same proliferation inhibitory influences of contact with stroma. Since the absolute number of primitive LTC-IC (week 8 LTC-IC) per mL of G-CSF mobilized PB is similar to that per mL of steady state BM, these studies further confirm that G-CSF mobilized PBPC may have similar long-term repopulating abilities as steady state BM.
Clonal Proliferation and Cytokine Requirement of Murine Progenitors for Natural Killer Cells
Authors: Aiba Yuichi , Hirayama Fumiya , Ogawa Makio
Published: October 2019

We have established a clonal cell culture system that supports the proliferation of committed natural killer (NK) cell progenitors of mice to investigate the pathway and cytokine regulation of NK cell development. Day 14 fetal thymocytes cultured in methylcellulose with interleukin-7 (IL-7), IL-15, and steel factor (SF ) formed diffuse colonies that could not be classified to known colony types. Single-cell origin of the colonies was established by micromanipulation of the colony-forming cells. Cells in the colonies are very blastic, showing no cytoplasmic differentiation, and express Ly5, Thy-1, and CD25 but not myeloid, B, mature T, or NK cell markers. The cells lack T, B, and myeloid potentials but can differentiate to mature NK cells in fetal thymus organ culture, suggesting that the colonies consist of NK committed progenitors. Examination of the minimal cytokine requirement for the NK colony formation showed that IL-7 and SF are indispensable for the formation of immature NK cell colonies. Both IL-2 and IL-15 increased the frequency of colonies. In contrast to IL-2, IL-7, and IL-15, IL-4 strongly inhibited the formation of the colonies. This quantitative clonal culture will provide a useful means to examine the mechanism of NK cell development.
Potential and Distribution of Transplanted Hematopoietic Stem Cells in a Nonablated Mouse Model
Authors: Nilsson Susan K. , Dooner Mark S. , Tiarks Candi Y. , Weier Heinz-Ulrich , Quesenberry Peter J.
Published: October 2019

Increasingly, allogeneic and even more often autologous bone marrow transplants are being done to correct a wide variety of diseases. In addition, autologous marrow transplants potentially provide an opportune means of delivering genes in transfected, engrafting stem cells. However, despite its widespread clinical use and promising gene therapy applications, relatively little is known about the mechanisms of engraftment in marrow transplant recipients. This is especially so in the nonablated recipient setting. Our data show that purified lineage negative rhodamine 123/Hoechst 33342 dull transplanted hematopoietic stem cells engraft into the marrow of nonablated syngeneic recipients. These cells have multilineage potential, and maintain a distinct subpopulation with “stem cell” characteristics. The data also suggests a spatial localization of stem cell “niches” to the endosteal surface, with all donor cells having a high spatial affinity to this area. However, the level of stem cell engraftment observed following a transplant of “stem cells” was significantly lower than that expected following a transplant of the same number of unseparated marrow cells from which the purified cells were derived, suggesting the existence of a “nonstem cell facilitator population,” which is required in a nonablated syngeneic transplant setting.
In Vitro Development of Erythroid and Megakaryocytic Cells From a UT-7 Subline, UT-7/GM
Authors: Komatsu Norio , Kirito Keita , Shimizu Ritsuko , Kunitama Masae , Yamada Minami , Uchida Mie , Takatoku Masaaki , Eguchi Mituoki , Miura Yasusada
Published: October 2019

UT-7 is a human megakaryoblastic leukemia cell line with absolute dependence on interleukin-3, granulocyte-macrophage colony-stimulating factor (GM-CSF ), or erythropoietin (EPO) for growth and survival. We isolated a novel subline, UT-7/GM after long-term culture of UT-7 with GM-CSF. The hemoglobin concentration and γ-globin and EPO-receptor mRNA levels were significantly higher in EPO-treated UT-7/GM cells than in untreated cells. In contrast, the platelet factor 4 and glycoprotein IIb mRNA levels were much higher in thrombopoietin (TPO)-treated UT-7/GM cells than in untreated cells. Some TPO-treated cells had morphologically mature megakaryocytic characteristics such as a developed demarcation membrane in the cytoplasm and multilobular nuclei. These findings indicate that UT-7/GM is a bipotential cell line that can be induced to differentiate into erythroid and megakaryocytic lineages by EPO and TPO, respectively. Moreover, a minority of UT-7/GM cells acquired a high hemoglobin concentration by treatment with TPO, suggesting that TPO in part induced the erythroid differentiation of the UT-7/GM cells. Interestingly, GM-CSF inhibited the EPO- or TPO-induced erythroid differentiation and the TPO-induced megakaryocytic differentiation of UT-7/GM cells. These results support the hypothesis that cytokines influence the programming of gene expression required for lineage commitment or differentiation.
Retroviral Gene Transduction of Adult Peripheral Blood or Marrow-Derived CD34+ Cells for Six Hours Without Growth Factors or on Autologous Stroma Does Not Improve Marking Efficiency Assessed In Vivo
Authors: Emmons R.V.B. , Doren S. , Zujewski J. , Cottler-Fox M. , Carter C.S. , Hines K. , O'Shaughnessy J.A. , Leitman S.F. , Greenblatt J.J. , Cowan K. , Dunbar C.E.
Published: October 2019

Our previous work in patients undergoing autologous transplant for multiple myeloma (MM) or breast cancer (BC) has shown that retroviral transduction of adult CD34+ cells for 72 hours in the presence of interleukin-3 (IL-3), IL-6, and stem cell factor (SCF ) resulted in .01% to 1% long-term marking of peripheral blood and marrow cells (Blood 85:3948, 1995). In this study we compare these previous studies to transduction with no added growth factors, previously shown to result in higher levels of marking in children (Lancet 342:1134, 1993) or transduction in the presence of an autologous stromal layer. Peripheral blood (PB) mononuclear cells were collected via apheresis after high-dose cyclophosphamide and granulocyte colony-stimulating factor. Bone marrow (BM) was also harvested in all patients. One third of both BM and PB collections were enriched for CD34+ cells and transduced with one of two marking vectors containing the neomycin-resistance gene to distinguish cells originating from BM and PB posttransplantation. Cells from 3 MM and 2 BC patients were transduced without growth factors for 6 hours and cells from 2 MM and 2 BC patients were transduced in the presence of autologous marrow stroma. Immediately posttransduction, the percentage of Neo-resistant PB and BM progenitors (colony-forming units) were: 0% to 19% in the 6-hour no growth factor group and 0% to 36% in the autologous stroma group. After conditioning therapy, both transduced and untransduced PB and BM fractions were infused into the patients. Semi-quantitative nested DNA polymerase chain reaction was performed on total, mononuclear, and granulocyte fractions of PB and BM at 1, 3, 6, 9, 12, and 18 months. Poor marking has been observed in both groups, with no consistently positive patients. These results compare unfavorably with our prior experience using growth factors during transduction. Further optimization of transduction conditions and vectors needs to be developed to improve transduction efficiency of adult human repopulating hematopoietic cells.
Mechanisms Responsible for Granzyme B–Independent Cytotoxicity
Authors: Shresta Sujan , Russell John H. , Ley Timothy J.
Published: October 2019

Using granzyme B–deficient mice obtained by gene targeting, we previously demonstrated that granzyme B is required for the rapid induction of apoptotic target cell death by cytotoxic T lymphocytes (CTLs); however, CTLs are also equipped with additional effector mechanisms. In the present study, we examined the mechanisms responsible for granzyme B–independent cytotoxicity using in vitro lytic assays with CTLs derived from mice deficient for both granzyme B and Fas ligand (FasL) (granzyme B−/− × gld/gld) or for perforin and FasL (perforin × gld/gld). Our results show that primary mixed lymphocyte reaction (MLR)-derived CTLs from granzyme B−/− × gld/gld mice induce apoptosis of allogeneic targets with less efficiency and a longer delay than CTLs deficient for granzyme B alone. The residual cytotoxicity in granzyme B−/− × gld/gld CTLs is primarily accounted for by a perforin-dependent mechanism, since perforin−/− × gld/gld CTLs have virtually no residual cytotoxic activity in our assays. Granzyme B–independent cytotoxicity is therefore partially accounted for by the Fas pathway and partially by another perforin-dependent mechanism.
Regulation of Interleukin-10 Gene Expression: Possible Mechanisms Accounting for Its Upregulation and for Maturational Differences in Its Expression by Blood Mononuclear Cells
Authors: Le Trong , Leung Ling , Carroll William L. , Schibler Kurt R.
Published: October 2019

Interleukin-10 (IL-10) downmodulates phagocytic immune responses and accentuates humoral responses. Human neonates exhibit broad immune deficits that parallel actions of IL-10. We postulated that IL-10 production would be diminished in neonatal blood cells. We found that IL-10 production by lipopolysaccharide-stimulated peripheral blood mononuclear cells (PBMNCs) in vitro was greater by adult cells than by term cells and preterm cells. Additional studies were undertaken to identify mechanisms responsible for the developmental differences in IL-10 gene expression. IL-10 transcription was present in freshly isolated adult and neonatal cells in the absence of detectable levels of transcript. Transcription rates were not different between adult and neonatal cells. IL-10 transcripts were approximately 40% more abundant in adult cells than in term cells and were consistent with differences in secreted protein; however, no differences were noted in mRNA stability. IL-10 half-life was 60 minutes for both adult and term PBMNCs. We conclude that up-regulation of IL-10 gene expression in PBMNCs is modulated at the post-transcriptional level, that IL-10 protein production and mRNA content are greater in activated cells from adults compared with those from neonates, and that maturational differences in IL-10 expression are not due to differences in transcription rate or mRNA stability. Maturational differences in IL-10 expression might be due to differences in subpopulations of cytokine-producing cells or differences in nucleo-cytoplasmic transport.
Tumor Necrosis Factor Constellation Polymorphism and Clozapine-Induced Agranulocytosis in Two Different Ethnic Groups
Authors: Turbay David , Lieberman Jeffrey , Alper Chester A. , Delgado Julio C. , Corzo Deyanira , Yunis Juan J. , Yunis Edmond J.
Published: October 2019

Genes of the major histocompatibility complex (MHC) are associated with susceptibility to different immune and nonimmune mediated diseases. We had reported that the drug adverse reaction, clozapine-induced agranulocytosis (CA), is associated with different HLA types and HSP70 variants in Ashkenazi Jewish and non-Jewish patients, suggesting that a gene within the MHC region is associated with CA. This study was designed to find common genetic markers for this disorder in both ethnic groups. The tumor necrosis factor (TNF ) microsatellites d3 and b4 were found in higher frequencies in both Jewish and nonJewish patients: 51 of 66 (77%) and 48 of 66 (57%), respectively. Comparisons of these frequencies with those of controls, 28 of 66 (42%) and 18 of 66 (27%), were statistically significant (corrected P value = .001 for the d3 allele and .0005 for the b4 allele). On the other hand, the TNF microsatellite b5 was underrepresented in the group of patients, 9 of 66 (14%), when compared with the control subjects, 43 of 66 (65%) (corrected P value = .0005), probably related to protection from CA. Our results show a strong association of some genetic variants of the TNF loci with susceptibility to CA in two different ethnic groups suggesting involvement of TNF and/or associated gene(s) products in the pathogenesis of this hematologic-drug adverse reaction.
Human Erythrocytes Express GLUT5 and Transport Fructose
Authors: Concha Ilona I. , Velásquez Fernando V. , Martı́nez Juan M. , Angulo Constanza , Droppelmann Andrea , Reyes Alejandro M. , Slebe Juan Carlos , Vera Juan Carlos , Golde David W.
Published: October 2019

Although erythrocytes readily metabolize fructose, it has not been known how this sugar gains entry to the red blood cell. We present evidence indicating that human erythrocytes express the fructose transporter GLUT5, which is the major means for transporting fructose into the cell. Immunoblotting and immunolocalization experiments identified the presence of GLUT1 and GLUT5 as the main facilitative hexose transporters expressed in human erythrocytes, with GLUT2 present in lower amounts. Functional studies allowed the identification of two transporters with different kinetic properties involved in the transport of fructose in human erythrocytes. The predominant transporter (GLUT5) showed an apparent Km for fructose of approximately 10 mmol/L. Transport of low concentrations of fructose was not affected by 2-deoxy–D-glucose, a glucose analog that is transported by GLUT1 and GLUT2. Similarly, cytochalasin B, a potent inhibitor of the functional activity of GLUT1 and GLUT2, did not affect the transport of fructose in human erythrocytes. The functional properties of the fructose transporter present in human erythrocytes are consistent with a central role for GLUT5 as the physiological transporter of fructose in these cells.
A Transgenic Mouse Model of Hemoglobin S Antilles Disease
Authors: Popp R.A. , Popp D.M. , Shinpock S.G. , Yang M.Y. , Mural J.G. , Aguinaga M.d.P. , Kopsombut P. , Roa P.D. , Turner E.A. , Rubin E.M.
Published: October 2019

Hemoglobin (Hb) S Antilles is a naturally occurring form of sickling human Hb but causes a more severe phenotype than Hb S. Two homozygous viable Hb S Antilles transgene insertions from Tg58Ru and Tg98Ru mice were bred into MHOAH mice that express high oxygen affinity (P50 ∼24.5 mm Hg) rather than normal (P50 ∼40 mm Hg) mouse Hbs. The rationale was that the high oxygen affinity MHOAH Hb, the lower oxygen affinity of Hb S Antilles than Hb S (P50 ∼40 v 26.5 mm Hg), and the lower solubility of deoxygenated Hb S Antilles than Hb S (∼11 v 18 g/dL) would favor deoxygenation and polymerization of human Hb S Antilles in MHOAH mouse red blood cells (RBCs). The Tg58 × Tg98 mice produced have a high and balanced expression (∼50% each) of hα and hβS Antilles globins, 25% to 35% of their RBCs are misshapen in vivo, and in vitro deoxygenation of their blood induces 30% to 50% of the RBCs to form classical looking, elongated sickle cells with pointed ends. Tg58 × Tg98 mice exhibit reticulocytosis, an elevated white blood cell count and lung and kidney pathology commonly found in sickle cell patients, which should make these mice useful for experimental studies on possible therapeutic intervention of sickle cell disease.
Use of Domain-Deletion Mutants to Locate Lutheran Blood Group Antigens to Each of the Five Immunoglobulin Superfamily Domains of the Lutheran Glycoprotein: Elucidation of the Molecular Basis of the Lua/Lub and the Aua/Aub Polymorphisms
Authors: Parsons S.F. , Mallinson G. , Daniels G.L. , Green C.A. , Smythe J.S. , Anstee D.J.
Published: October 2019

Lutheran glycoprotein (Lu gp) has five predicted immunoglobulin superfamily (IgSF ) domains. K562 cells were transfected with Lu cDNA and tested by flow cytometry with monoclonal antibodies and Lu blood group antisera. The results confirmed the identity of Lu cDNA. Deletion mutants lacking the regions encoding one or more IgSF domains were made by inverse polymerase chain reaction (PCR), expressed in K562 cells, and tested with the same antibodies. The Lub and Lu5 antigens and the epitope recognized by monoclonal antibody BRIC 224 were mapped to the first, N-terminal, IgSF domain. Lu4 and Lu8 were mapped to domain 2; Lu20 to domain 3; Lu7 and BRIC 221 epitope to domain 4, and Lu13 and Aub to domain 5. The organization of the LU gene was determined. The region encoding the open reading frame is arranged in 15 exons extending over ≈11 kb on chromosome 19q13.2. The Lua/Lub and Aua/Aub blood group polymorphisms were studied using genomic DNA from typed blood donors. The Lua mutation is a base change in exon 3 (G252 to A) encoding an Arg77 (Lub) to His (Lua) change on the CFG face of domain 1. The Aua/Aub polymorphism is an A1637 to G substitution in exon 12 encoding a Thr539 (Aua) to Ala (Aub) change on the G strand of domain 5.
Molecular Cloning and Characterization of a cDNA, CHEMR1, Encoding a Chemokine Receptor With a Homology to the Human C-C Chemokine Receptor, CCR-4
Authors: Youn Byung-S. , Kim Sang-Ho , Lyu Myung S. , Kozak Christine A. , Taub Dennis D. , Kwon Byoung S.
Published: October 2019

Chemokines refer to a rapidly expanding family of small cytokines whose primary function is recruitment of leukocytes to inflammatory sites. These are known to bind to seven-transmembrane-domain containing receptors. A cDNA clone, CHEMR1, resembling the typical G protein-coupled receptor, was isolated from a mouse cytotoxic T-lymphocyte (CTL) library. Northern blot analysis in mouse cell lines suggests that its expression is found in a variety of cells, including T cells, B cells, and macrophages. The CHEMR1 gene Scya3r2 is a single-copy gene whose open reading frame may be in a single exon and maps to the distal region of mouse Chr 9 where the mouse macrophage inflammatory protein-1α (MIP-1α) receptor gene Scya3r and two related C-C chemokine receptor-like genes reside. Amino acid sequence comparison shows that CHEMR1 is 84% identical to human CCR-4, indicating that CHEMR1 is likely to be a mouse CCR-4. Binding assays using 125I-labeled C-C chemokines in mammalian cells indicated that CHEMR1 did not bind MIP-1α, RANTES, or MIP-1β, whereas CCR-1 binds MIP-1α and RANTES. Our result is different from the reported properties of human CCR-4. This suggests that CHEMR1 may be a receptor for unidentified C-C chemokine or a low-affinity receptor for MIP-1α.
Interleukin-10 Inhibits Interferon-γ–Induced Intercellular Adhesion Molecule-1 Gene Transcription in Human Monocytes
Authors: Song Seng , Ling-Hu Hsiang , Roebuck Kenneth A. , Rabbi Mohammed F. , Donnelly Raymond P. , Finnegan Alison
Published: October 2019

Interleukin-10 (IL-10) is a potent monocyte regulatory cytokine that inhibits gene expression of proinflammatory mediators. In this study, we investigated the mechanism by which IL-10 downregulates expression of intercellular adhesion molecule-1 (ICAM-1) on the cell surface of normal human monocytes activated with interferon-γ (IFN-γ). IL-10 inhibition of IFN-γ–induced ICAM-1 expression was apparent as early as 3 hours and was blocked by an anti–IL-10 antibody but not by an isotype-matched control antibody. Northern blot analysis showed that IL-10 reduced the accumulation of ICAM-1 mRNA in IFN-γ–stimulated monocytes. IL-10 inhibition of ICAM-1 steady-state mRNA was detected at 3 hours and remained at 24 hours. Nuclear run-on transcription assays showed that IL-10 inhibited the rate of IFN-γ–induced transcription of the ICAM-1 gene, and mRNA stability studies showed that IL-10 did not alter the half-life of IFN-γ–induced ICAM-1 message. Thus, IL-10 inhibits IFN-γ–induced ICAM-1 expression in monocytes primarily at the level of gene transcription. Activation of IFN-γ–responsive genes requires tyrosine phosphorylation of the transcriptional factor STAT-1α (signal transducer and activator of transcription-1α). However, IL-10 did not affect IFN-γ–induced tyrosine phosphorylation of STAT-1α or alter STAT-1α binding to the IFN-γ response element (IRE) in the ICAM-1 promoter. Instead, IL-10 prevented IFN-γ–induced binding activity at the NF-κB site of the tumor necrosis factor α (TNF-α)–responsive NF-κB/C-EBP composite element in the ICAM-1 promoter. These data indicate that IL-10 inhibits IFN-γ–induced transcription of the ICAM-1 gene by a regulatory mechanism that may involve NF-κB.
Nonnasal Lymphoma Expressing the Natural Killer Cell Marker CD56: A Clinicopathologic Study of 49 Cases of an Uncommon Aggressive Neoplasm
Authors: Chan John K.C. , Sin V.C. , Wong K.F. , Ng C.S. , Tsang William Y.W. , Chan C.H. , Cheung M.M.C. , Lau W.H.
Published: October 2019

Expression of the natural killer (NK) cell antigen CD56 is uncommon among lymphomas, and those that do are almost exclusively of non–B-cell lineage and show a predilection for the nasal and nasopharyngeal region. This study analyzes 49 cases of nonnasal CD56+ lymphomas, the largest series to date, to characterize the clinicopathologic spectrum of these rare neoplasms. All patients were Chinese. Four categories could be delineated. (1) Nasal-type NK/T cell lymphoma (n = 34) patients were adults 21 to 76 years of age (median, 50 years), including 25 men and 9 women. They presented with extranodal disease, usually in multiple sites. The commonest sites of involvement were skin, upper aerodigestive tract, testis, soft tissue, gastrointestinal tract, and spleen. Only 7 cases (21%) apparently had stage I disease. The neoplastic cells were often pleomorphic, with irregular nuclei and granular chromatin, and angiocentric growth was common. The characteristic immunophenotype was CD2+ CD3/Leu4− CD3ε+ CD56+, and 32 cases (94%) harbored Epstein-Barr virus (EBV). Follow-up information was available in 29 cases: 24 died at a median of 3.5 months; 3 were alive with relapse at 5 months to 2.5 years; and 2 were alive and well at 3 and 5 years, respectively. (2) Aggressive NK cell leukemia/lymphoma (n = 5) patients presented with hepatomegaly and blood/marrow involvement, sometimes accompanied by splenomegaly or lymphadenopathy. The neoplastic cells often had round nuclei and azurophilic granules in the pale cytoplasm. All cases exhibited an immunophenotype of CD2+ CD3/Leu4− CD56+ CD16− CD57− and all were EBV+. All of these patients died within 6 weeks. (3) In blastoid NK cell lymphoma (n = 2), the lymphoma cells resembled those of lymphoblastic or myeloid leukemia. One case studied for CD2 was negative and both cases were EBV−. One patient was alive with disease at 10 months and one was a recent case. (4) Other specific lymphoma types with CD56 expression (n = 8) included one case each of hepatosplenic γδ T-cell lymphoma and S100 protein+ T-cell lymphoproliferative disease and two cases each of T-chronic lymphocytic/prolymphocytic leukemia, lymphoblastic lymphoma, and true histiocytic lymphoma. All of these cases were EBV−. Six patients died at a median of 6.5 months. Nonnasal CD56+ lymphomas are heterogeneous, but all pursue a highly aggressive clinical course. The nasal-type NK/T-cell lymphoma and aggressive NK cell leukemia/lymphoma show distinctive clinicopathologic features and a very strong association with EBV. Blastoid NK cell lymphoma appears to be a different entity and shows no association with EBV.
Plasmin Is a Potent and Specific Chemoattractant for Human Peripheral Monocytes Acting Via a Cyclic Guanosine Monophosphate–Dependent Pathway
Authors: Syrovets Tatjana , Tippler Bettina , Rieks Maike , Simmet Thomas
Published: October 2019

We have previously reported that the serine protease plasmin generated during contact activation of human plasma triggers biosynthesis of leukotrienes (LTs) in human peripheral monocytes (PMs), but not in polymorphonuclear neutrophils (PMNs). We now show that purified plasmin acts as a potent chemoattractant on human monocytes, but not on PMNs. Human plasmin or plasminogen activated with urokinase, but not active site-blocked plasmin or plasminogen, elicited monocyte migration across polycarbonate membranes. Similarly, stimulation of monocytes with plasmin, but not with active site-blocked plasmin or plasminogen, induced actin polymerization. As assessed by checkerboard analysis, the plasmin-mediated monocyte locomotion was a true chemotaxis. The plasmin-induced chemotactic response was inhibited by the lysine analog trans-4-(aminomethyl)cyclohexane-1-carboxylic acid (t-AMCA), which prevents binding of plasmin/ogen to the appropriate membrane binding sites. In addition, active site-blocked plasmin inhibited monocyte migration triggered by active plasmin. Further, plasmin-induced monocyte chemotaxis was inhibited by pertussis toxin (PTX) and 1-O-hexadecyl-2-O-methyl-rac-glycerol (HMG) and chelerythrine, two structurally unrelated inhibitors of protein kinase C (PKC). Plasmin, but not active site-blocked plasmin or plasminogen, triggered formation of cyclic guanosine monophosphate (cGMP) in monocytes. LY83583, an inhibitor of soluble guanylyl cyclase, inhibited both plasmin-induced cGMP formation and the chemotactic response. The latter effect could be antagonized by 8-bromo-cGMP. In addition, KT5823 and (Rp)-8-(p-chlorophenylthio)guanosine-3′,5′-cyclic monophosphorothioate [(Rp)-8-pCPT-cGMPs], two structurally unrelated inhibitors of cGMP-dependent protein kinase, inhibited plasmin-mediated monocyte chemotaxis. Thus, beyond being a stimulus for lipid mediator release, plasmin is a potent and specific chemoattractant for human monocytes acting via a cGMP-dependent mechanism. Therefore, plasmin represents a proinflammatory activator for human monocytes.
Familial Erythrocytosis Associated With a Short Deletion in the Erythropoietin Receptor Gene
Authors: Arcasoy Murat O. , Degar Barbara A. , Harris Kevin W. , Forget Bernard G.
Published: October 2019

Familial erythrocytosis (familial polycythemia) inherited as an autosomal dominant trait has recently been reported to be associated with mutations in the gene encoding the erythropoietin receptor (EpoR) in a small number of families. We studied a new kindred with dominantly inherited familial erythrocytosis associated with heterozygosity for a deletion of seven nucleotides between positions 5985 and 5991 in exon 8 of the EpoR gene, resulting in an EpoR peptide that is truncated by 59 amino acids at its C-terminus. A 7-bp direct repeat is present in the normal EpoR gene at the site of this mutation, consistent with the slipped mispairing model for the generation of short deletions during DNA replication. Hypersensitivity to Epo of erythroid progenitors from an affected individual was observed in in vitro methylcellulose cultures, as indicated by more numerous and larger colonies compared with those of a control subject. To study mutant EpoR function, the cDNA encoding the mutant EpoR was synthesized by reverse transcription-polymerase chain reaction of peripheral blood RNA from the proband and stably tranfected into murine interleukin-3–dependent 32D cells. Epo dose-response assays showed that cells expressing the mutant EpoR displayed fivefold to 10-fold increased sensitivity to Epo compared with cells expressing similar numbers of the wild-type EpoR.
A Phase-I Study of an Anti-CD25 Ricin A-Chain Immunotoxin (RFT5-SMPT-dgA) in Patients With Refractory Hodgkin's Lymphoma
Authors: Engert Andreas , Diehl Volker , Schnell Roland , Radszuhn Andrea , Hatwig Maria-Theresia , Drillich Silke , Schön Gisela , Bohlen Heribert , Tesch Hans , Hansmann Martin-Leo , Barth Stefan , Schindler John , Ghetie Victor , Uhr Jonathan , Vitetta Ellen
Published: October 2019

The anti-CD25 immunotoxin (IT), RFT5-SMPT-dgA, was used in a phase I dose escalation trial in patients with refractory Hodgkin's lymphoma. The IT was constructed by linking the monoclonal antibody RFT5 via a sterically hindered disulfide linker to deglycosylated ricin-A. All patients in this trial were heavily pretreated with a mean of 5 (range, 2 to 8) different prior therapies, including autologous bone marrow transplantation in 8 of 15. The mean age was 29 years (range, 19 to 34 years). Thirteen of 15 patients had advanced disease (stage IV) with massive tumor burdens and 6 of 15 had B symptoms. The IT was administered intravenously over 4 hours on days 1, 3, 5, and 7 for total doses per cycle of 5, 10, 15, or 20 mg/m2. Patients received one to four cycles of treatment. The peak serum concentration of intact IT varied from 0.2 to 9.7 μg/mL. The serum half life (T1/2 ) of the IT ranged from 4.0 to 10.5 hours (mean, 6.1 hours). Side effects were related to vascular leak syndrome (VLS), ie, decreases in serum albumin, edema, weight gain, hypotension, tachycardia, myalgia, and weakness. Two patients had a National Cancer Institute (NCI) grade 2 allergic reaction with generalized urticaria and mild bronchospasm. At 15 mg/m2, 1 patient experienced a grade 3 myalgia. All 3 patients receiving 20 mg/m2 experienced NCI grade 3 toxicities (edema, nausea, dyspnea or tachycardia) and 1 patient had NCI grade 4 myalgia. Thus, the maximal tolerated dose was 15 mg/m2. Seven of 15 patients made human antiricin antibodies (≥1.0 μg/mL) and 6 of 15 developed human antimouse antibodies (≥1.0 μg/mL). Clinical response included 2 partial remissions, 1 minor response, 3 stable diseases, and 9 progressive diseases. As has been predicted from the preclinical tests, these data seem to indicate clinical effecicacy of this new IT in heavily pretreated Hodgkin's patients, thus warranting further clinical investigation.
Avoidance of Allogeneic Blood Transfusions by Treatment With Epoetin Beta (Recombinant Human Erythropoietin) in Patients Undergoing Open-Heart Surgery
Authors: Sowade Olaf , Warnke Harry , Scigalla Paul , Sowade Birgit , Franke Werner , Messinger Diethelm , Gross Johann
Published: October 2019

In a double-blind, randomized, placebo-controlled trial, we evaluated the ability of epoetin beta (recombinant human erythropoietin) to avoid allogeneic blood transfusions (ABT) and the associated risks in patients undergoing primary elective open-heart surgery and in whom autologous blood donation (ABD) was contraindicated. Seventy-six patients overall were enrolled onto the trial and were randomly assigned to the two treatment groups, 5 × 500 U/kg body weight (BW) epoetin beta or placebo intravenously over 14 days preoperatively. All patients received 300 mg Fe2+ orally per day during the treatment period. Preoperatively, the mean hemoglobin increase was 1.50 g/dL greater in epoetin beta patients than in placebo patients (95% confidence interval, 1.10 to 1.90 g/dL), allowing a rapid return to the baseline value by the seventh postoperative day in most epoetin beta patients. The mean volume of blood collected by intraoperative isovolemic hemodilution was 562 mL (red blood cell mass, 274 mL) in the epoetin beta group and 218 mL (red blood cell mass, 94 mL) in the placebo group, respectively. Only four patients (11%) in the epoetin beta group received an ABT, compared with 19 (53%) in the placebo group (P = .0003). Epoetin beta was most useful in patients with a perioperative blood loss greater than 750 mL, in those with a baseline hematocrit value less than 0.42, and in those aged ≥60 years. The iron supplementation proved adequate despite the fact that a significant decrease in ferritin (median, 48.1%) and transferrin saturation (median, 40.5%) was observed in epoetin beta patients preoperatively. No influence of epoetin beta therapy on blood pressure, laboratory safety variables, or the frequency of specific adverse events was observed. Intravenous epoetin beta treatment of 5 × 500 U/kg BW in combination with 300 mg Fe2+ orally per day administered over 14 days preoperatively is an adequate therapy for increasing mean hemoglobin levels by approximately 1.50 g/dL and reducing the allogeneic blood requirement in patients undergoing elective open-heart surgery and in whom ABD is contraindicated.
3′-Azido 3′-Deoxythymidine + Methotrexate as a Novel Antineoplastic Combination in the Treatment of Human Immunodeficiency Virus-Related Non-Hodgkin's Lymphomas
Authors: Tosi Patrizia , Gherlinzoni Filippo , Mazza Patrizio , Visani Giuseppe , Coronado Olga , Costigliola Paolo , Raise Enzo , Mazzetti Magda , Gritti Francesco , Chiodo Francesco , Tura Sante
Published: October 2019

We have previously reported that 3′-azido 3′-deoxythymidine (AZT) can possess a significant antineoplastic activity when combined with drugs that disrupt de novo thymidylate synthesis, such as 5-fluorouracil and methotrexate (MTX). The aim of the present study was to evaluate the efficacy and the tolerance of the combination AZT + MTX in human immunodeficiency virus (HIV)-related non-Hodgkin's lymphoma (NHL). Twenty-nine patients (22 men and 7 women), either newly diagnosed or pretreated, have been enrolled in the trial; the median age was 34 years, 45% had acquired immunodeficiency syndrome before lymphoma and 19 patients had less than 100 CD4 lymphocytes/μL. Histologic diagnoses were mainly Burkitt (27%) and diffuse large B-cell lymphoma (45%); extranodal involvement was present in 20 patients. The treatment plan included three weekly courses of MTX at 1 g/m2 (days 1, 8, and 15) plus oral AZT at 2 g/m2 (days 1, 2, and 3), 4 g/m2 (days 8, 9, and 10), and 6 g/m2 (days 15, 16, and 17), plus leucovorin rescue. From the eleventh patient on, in case of complete or partial remission, the treatment was continued with three additional courses, using AZT at the maximum dose. In 26 evaluable patients, the total (complete + partial) response rate was 77% (95% confidence interval, 58% to 89%), with complete remission (CR) in 46% of the patients (95% confidence interval, 29% to 65%). The median CR duration was 12.8 months. Grade III-IV neutropenia and anemia were observed in 52% and 31% of the courses, respectively. There was one therapy-related death due to bacteremia followed by septic shock; the only other recorded infection was a herpes vaginalis. In conclusion, we suggest that AZT + MTX is an effective and well-tolerated regimen in HIV-related NHL.
Thrombopoietin Signal Transduction in Purified Murine Megakaryocytes
Authors: Drachman Jonathan G. , Sabath Diana F. , Fox Norma E. , Kaushansky Kenneth
Published: October 2019

Thrombopoietin (TPO) is a recently cloned cytokine that binds to its receptor, Mpl, and promotes hematopoietic expansion and maturation, primarily of the megakaryocyte lineage. The signaling pathways responsible for these events are thought to involve the Janus family of nonreceptor tyrosine kinases (JAKs) and the signal transducers and activators of transcription (STATs), which are activated by tyrosine phosphorylation. Previous investigators have studied these molecules in engineered and naturally occurring cell lines. To investigate the molecular basis for TPO signal transduction in a more physiologic target, we determined the pattern of JAK and STAT activation in purified, normal murine megakaryocytes. These results are compared with those of established cell lines that only proliferate (Ba/F3-mMPL and DA-1-TPO) or only differentiate (L8057) in response to TPO. From these findings, a model is proposed to explain the physiologic roles of JAK2, TYK2, STAT3, and STAT5 in TPO signaling. Furthermore, previous studies of the physical interaction between Mpl and the JAKs are extended, showing a difference in the association of JAK2 and TYK2 with the TPO receptor. Finally, we show that, in the cell line Ba/F3-mMPL, the closely related proteins STAT5A and STAT5B are both activated by TPO stimulation and are capable of heterodimerization. Together, these results further our understanding of the early stages of megakaryocyte and platelet development.
p15s (15-kD Antimicrobial Proteins) Are Stored in the Secondary Granules of Rabbit Granulocytes: Implications for Antibacterial Synergy With the Bactericidal/Permeability-Increasing Protein in Inflammatory Fluids
Authors: Zarember Kol , Elsbach Peter , Shin-Kim Kwang , Weiss Jerrold
Published: October 2019

The bactericidal potency toward complement-resistant Escherichia coli of bactericidal/permeability-increasing protein (BPI) released from polymorphonuclear leukocytes (PMNs) in glycogen-induced inflammatory peritoneal exudates of rabbits is dependent on synergy with extracellular p15s. This synergy depends on the high molar ratio of p15s to BPI in the extracellular fluid (∼50:1), which greatly exceeds the intracellular ratio (∼5:1). To explore the possible basis of the greater accumulation of p15s in inflammatory fluid, we examined the subcellular localization of BPI and p15 in PMNs. Immunogold electron microscopy confirmed the storage of BPI in primary granules and showed that p15s are stored in secondary granules. Reverse-transcription polymerase chain reaction of density-fractionated rabbit bone marrow cells verified that p15s are expressed later than BPI during myeloid differentiation. As the inflammatory response evolves, p15 mRNA appears earlier in blood and exudate cells than mRNA for BPI, consistent with release of progressively less mature precursors from bone marrow. Finally, Ca2+-ionophore–mediated exocytosis of p15s occurs more readily than release of BPI. We therefore propose that localization of a synergistic partner of BPI (p15s) in more readily released secondary granules allows the neutrophil to mobilize potent BPI-dependent antibacterial activity extracellularly without significant depletion of intracellular BPI stores.
Granulocyte-Macrophage Colony-Stimulating Factor–Activated Signaling Pathways in Human Neutrophils. I. Tyrosine Phosphorylation-Dependent Stimulation of Phosphatidylinositol 3-Kinase and Inhibition by Phorbol Esters
Authors: Al-Shami Amin , Bourgoin Sylvain G. , Naccache Paul H.
Published: October 2019

Phosphatidylinositol 3-kinase (PI3-kinase) is a cytosolic enzyme that plays key roles in mediating signaling through many receptors. The heterodimeric form of PI3-kinase is made up of a regulatory subunit, p85, and a catalytic subunit, p110. Although granulocyte-macrophage colony-stimulating factor (GM-CSF) has been shown to activate PI3-kinase, the mechanisms by which this activation is mediated and regulated are incompletely understood. Here we show that treatment of human neutrophils with GM-CSF induced both time- and concentration-dependent increases in the level of tyrosine phosphorylation of p85. The ability of GM-CSF to activate PI3-kinase was abolished by pretreating the cells with erbstatin, a tyrosine kinase inhibitor. The simultaneous treatment of the cells with GM-CSF and phorbol esters such as phorbol 12-myristate 13-acetate (PMA) and phorbol 12,13-dibutyrate (PDBu) significantly inhibited both the tyrosine phosphorylation of p85 and the activation of PI3-kinase. The inhibitory effects of phorbol esters were not induced by their inactive analogues and they were selective to the stimulation of tyrosine phosphorylation of p85 since phorbol esters did not alter the enhancement of the pattern of tyrosine phosphorylation of other cellular proteins, including that of Jak2 induced by GM-CSF. However, PMA significantly inhibited the in situ tyrosine phosphorylation and the activation of lyn observed in response to GM-CSF. The results suggest that the activation of PI3-kinase by GM-CSF is mediated by the tyrosine phosphorylation of p85 and that this activation is downregulated by PKC possibly via the inhibition of lyn.
Serum Transferrin Receptor and Its Ratio to Serum Ferritin in the Diagnosis of Iron Deficiency
Authors: Punnonen Kari , Irjala Kerttu , Rajamäki Allan
Published: October 2019

The objective of the study was to evaluate the diagnostic efficiency of laboratory tests, including serum transferrin receptor (TfR) measurements, in the diagnosis of iron depletion. The patient population consisted of 129 consecutive anemic patients at the University Hospital of Turku who were given a bone marrow examination. Of these patients, 48 had iron deficiency anemia (IDA), 64 anemia of chronic disease (ACD), and 17 patients had depleted iron stores and an infectious or an inflammatory condition (COMBI). Depletion of iron stores was defined as a complete absence of stainable iron in the bone marrow examination. Serum TfR concentrations were elevated in the vast majority of the IDA and COMBI patients, while in the ACD patients, the levels were within the reference limits reported earlier for healthy subjects. TfR measurement thus provided a reliable diagnosis of iron deficiency anemia (AUCROC 0.98). Serum ferritin measurement also distinguished between IDA patients and ACD patients. However, the optimal decision limit for evaluation of ferritin measurements was considerably above the conventional lower reference limits, complicating the interpretation of this parameter. Calculation of the ratio TfR/log ferritin (TfR-F Index) is a way of combining TfR and ferritin results. This ratio provided an outstanding parameter for the identification of patients with depleted iron stores (AUCROC 1.00). In anemic patients, TfR measurement is a valuable noninvasive tool for the diagnosis of iron depletion, and offers an attractive alternative to more conventional laboratory tests in the detection of depleted iron stores.
High-Dose Therapy and Autologous Hematopoietic Progenitor Cell Transplantation for Recurrent or Refractory Hodgkin's Disease: Analysis of the Stanford University Results and Prognostic Indices
Authors: Horning Sandra J. , Chao Nelson J. , Negrin Robert S. , Hoppe Richard T. , Long Gwynn D. , Hu Wendy W. , Wong Ruby M. , Brown Byron W. , Blume Karl G.
Published: October 2019

One hundred nineteen patients with relapsed or refractory Hodgkin's disease (HD) received high-dose therapy followed by autologous hematopoietic progenitor cell transplantation. Three preparatory regimens, selected on the basis of prior therapy and pulmonary status, were employed. Twenty-six patients without a history of prior chest or pelvic irradiation were treated with fractionated total body irradiation, etoposide (VP) 60 mg/kg and cyclophosphamide (Cy) 100 mg/kg. Seventy-four patients received BCNU 15 mg/kg with identical doses of VP and Cy. A group of 19 patients with a limited diffusing capacity or history of pneumonitis received a novel high-dose regimen consisting of CCNU 15 mg/kg, VP 60 mg/kg and Cy 100 mg/kg. Twenty-nine patients (24%) had failed induction therapy and 35 (29%) had progressive HD within 1 year of initial chemotherapy. At 4 years actuarial survival was 52%, event-free survival was 48% and freedom from progression (FFP) was 62%. No significant differences were seen in survival data with the three preparatory regimens. Six patients died within 100 days of transplantation and 5 died at a later date of transplant-related complications. Secondary malignancies have developed in 6 patients, including myelodysplasia/leukemia in four patients and solid tumors in two patients. Regression analysis identified systemic symptoms at relapse, disseminated pulmonary or bone marrow disease at relapse and more than minimal disease at the time of transplantation as significant prognostic factors for overall and event-free survival and FFP. Patients with none of these factors enjoyed an 85% FFP at 4 years compared with 41% for patients with one or more unfavorable prognostic factors (P = .0001). Our results confirm the efficacy of high-dose therapy and autografting in recurrent or refractory HD. Although longer follow-up is necessary to address ultimate cure rates and toxicity, our data indicate that a desire to reduce late effects should drive future research efforts in favorable patients whereas new initiatives are needed for those with less favorable prognoses.
Role of bcl-2 in the Development of Lymphoid Cells From the Hematopoietic Stem Cell
Authors: Matsuzaki Yumi , Nakayama Kei-ichi , Nakayama Keiko , Tomita Takashi , Isoda Miu , Loh Dennis Y. , Nakauchi Hiromitsu
Published: October 2019

To investigate the role of bcl-2 in lymphohematopoiesis, a long-term bone marrow reconstitution system was established. Transplantation of 1,000 c-Kit+ Sca-1+ and lineage markers negative cells from bcl-2−/− mouse bone marrow resulted in long-term reconstitution of nonlymphoid cells. However, T cells were totally absent and B-lymphocyte development was severely impaired at a very early stage of differentiation in the chimeric mouse. On the other hand, transplantation of day 14 fetal liver cells from bcl-2−/− mice resulted in generation of both T and B cells in the recipient, albeit transiently. These data suggest that bcl-2 plays a critical role in the development of lymphoid progenitor cells from the hematopoietic stem cell (HSC), but is not essential for the development of nonlymphoid cells and the self-renewal of HSC. In addition, lymphopoiesis from fetal liver HSC appears to be less dependent on bcl-2 than adult bone marrow HSC.