bluuebunny/crossref_metadata_2025 huggingface dataset:
Recurrent Arterial Thrombosis Linked to Autoimmune Antibodies Enhancing von Willebrand Factor Binding to Platelets and Inducing FcγRII Receptor-Mediated Platelet Activation
Authors: Hoylaerts Marc F. , Thys Chantal , Arnout Jef , Vermylen Jos
Published: October 2019

A patient with a history of recurrent late fetal loss associated with multiple placental infarcts and cerebrovascular ischemia at the age of 36, followed a year later by a myocardial infarction, was referred for further investigation. Coronary angiography was normal. Antinuclear factor, lupus anticoagulant, anticardiolipin antibodies, and other thrombophilia parameters were negative, but there was moderate hyperthyroidism with positive thyroid peroxidase antibodies. Platelet numbers and von Willebrand factor (vWF) were normal. Her platelets showed spontaneous aggregation that disappeared with aspirin intake. However, aggregation still was induced by low levels of ristocetin (0.3 to 0.5 mg/mL). The low-dose ristocetin aggregation in patient platelet-rich plasma (PRP) was completely blocked by neutralizing antiglycoprotein Ib (GPIb) and anti-vWF antibodies. The monoclonal anti-FcγRII receptor antibody IV.3 inhibited partly, which suggests that PRP aggregation by low-dose ristocetin was elicited by vWF-immunoglobulin (Ig) complexes. Upon addition to washed human platelets, with vWF (10 μg/mL), purified patient Igs dose-dependently enhanced ristocetin (0.15 mg/mL)-induced aggregation between 0 and 500 μg/mL, an effect that disappeared again above 1 mg/mL. Aggregation was dependent on the vWF concentration and was blocked by IV.3 or neutralizing anti-GPIb or anti-vWF antibodies. The spontaneous aggregation of normal platelets resuspended in patient plasma could be inhibited totally by IV.3 and partially by neutralizing anti-GPIb or anti-vWF antibodies. Perfusion with normal anticoagulated blood, enriched with 10% of control or patient plasma, over surfaces coated with vWF showed increased platelet adhesion and activation in the presence of patient antibodies. Treatment of the patient with the antithyroid drug thiamazol and temporary corticosteroids, aspirin, and ticlopidine did not correct the platelet hypersensitivity to ristocetin. These observations suggest that some autoantibodies to vWF may both enhance vWF binding to platelets and cause platelet activation through binding to the FcγRII receptor, and thereby may be responsible for a new form of antibody-mediated thrombosis.
Macrophage Inflammatory Protein-1β Induces Migration and Activation of Human Thymocytes
Authors: Dairaghi Daniel J. , Franz-Bacon Karin , Callas Eleni , Cupp James , Schall Thomas J. , Tamraz Susan A. , Boehme Stefen A. , Taylor Naomi , Bacon Kevin B.
Published: October 2019

The CC chemokine macrophage inflammatory protein 1β (MIP-1β), has been shown to be a chemoattractant preferentially activating CD4+ CD45RA+ T lymphocytes. Further analysis of chemokine action on lymphocytic cells has shown the potent migration-promoting capacity of MIP-1β on human thymocytes. The responding cells were the CD4+ and CD8+single-positive (SP), as well as the CD4+CD8+ double-positive (DP) populations, with little if any migratory activity on the double-negative (DN) population. The activation of thymocytes by MIP-1β appeared to be a direct, receptor-mediated event as evidenced by the rapid mobilization of intracellular calcium, increase in proteins phosphorylated on tyrosine, and activation of the mitogen-activated protein kinase (MAPK) pathway. Radioligand binding analyses showed specific and displaceable binding of MIP-1β to thymocytes with a Kd of approximately 1 nmol/L, a profile that was comparable with MIP-1β binding to CCR-5–transfected NIH 3T3 cells. In addition, CCR-5 mRNA was detected in total thymocyte populations indicating that activation of thymocytes by MIP-1β may occur through binding to CCR-5. Further dissection of the subpopulations showed that only the DP and CD8+ SP populations expressed CCR-5 and expression data on these two populations was confirmed using anti–CCR-5 monoclonal antibody. These data may be suggestive of a role for MIP-1β in human thymocyte activation, and show a potential route for HIV infectivity in the developing immune system.
The Clinical Significance of Molecular Response in Indolent Follicular Lymphomas
Authors: López-Guillermo Armando , Cabanillas Fernando , McLaughlin Peter , Smith Terry , Hagemeister Fredrick , Rodrı́guez Marı́a A. , Romaguera Jorge E. , Younes Anas , Sarris Andreas H. , Preti H. Alejandro , Pugh William , Lee Ming-Seng
Published: October 2019

Most patients with follicular lymphoma (FL) achieve a complete response (CR) after treatment, but eventually most of them, particularly those with stage IV, relapse due to minimal residual disease (MRD). The t(14;18) gives rise to a rearrangement of the bcl-2 oncogene that constitutes an excellent target for detection of MRD by polymerase chain reaction (PCR). One hundred ninety-four previously untreated patients with indolent FL and detectable bcl-2 rearrangement were studied. The PCR assay was used to detect bcl-2–rearranged cells in blood and marrow before and after treatment. Molecular response rate was 37%, 53%, 56%, and 66% at 3 to 5, 6 to 8, 9 to 14, and 15 to 18 months from the start of therapy, respectively. Although molecular response was higher among clinical CRs, one third of partial responders at 3 to 5 months also achieved a molecular response. Patients who achieved a molecular response during the first year of treatment had a significantly longer failure-free survival (FFS) than those who did not (4-year FFS: 76% v 38%, respectively; P < .001). Similar results were also observed in the subset of patients in clinical CR 1 year after treatment. By multivariate analysis, β2-microglobulin (β2-M; P < .01), and molecular response (P < .001) were the most important variables associated with outcome. When we combined β2-M and molecular response, three prognostic groups emerged: (1) low β2-M and molecular responders, (2) low β2-M and nonresponders or high β2-M and responders, and (3) high β2-M and nonresponders. The 4-year FFS of these 3 groups were 86%, 65%, and 23%, respectively. Finally, patients who achieved molecular response and sustained it had better FFS than those who either reverted back to PCR-positive or who never achieved molecular response. Serial PCR analysis to determine the molecular response in FL correlates well with outcome especially when combined with pretreatment β2-M.
Wilms' Tumor Gene (WT1) Competes With Differentiation-Inducing Signal in Hematopoietic Progenitor Cells
Authors: Inoue Kazushi , Tamaki Hiroya , Ogawa Hiroyasu , Oka Yoshihiro , Soma Toshihiro , Tatekawa Toyoshi , Oji Yusuke , Tsuboi Akihiro , Kim Eui Ho , Kawakami Manabu , Akiyama Tetsu , Kishimoto Tadamitsu , Sugiyama Haruo
Published: October 2019

The WT1 gene is a tumor-suppressor gene that was isolated as a gene responsible for Wilms' tumor, a childhood kidney neoplasm. We have previously reported that the WT1 gene is strongly expressed in leukemia cells with an increase in its expression levels at relapse and an inverse correlation between its expression levels and prognosis, thus making it a novel tumor marker for leukemic blast cells. Furthermore, WT1 antisense oligomers have been found to inhibit the growth of leukemic cells. These results strongly suggested the involvement of the WT1 gene in human leukemogenesis. The present study was performed to prove our hypothesis that the WT1 gene plays a key role in leukemogenesis and performs an oncogenic function in hematopoietic progenitor cells, rather than a tumor-suppressor gene function. 32D cl3, an interleukin-3–dependent myeloid progenitor cell line, differentiates into mature neutrophils in response to granulocyte colony-stimulating factor (G-CSF). However, when transfected wild-type WT1 gene was constitutively expressed in 32D cl3, the cells stopped differentiating and continued to proliferate in response to G-CSF. As for signal transduction mediated by G-CSF receptor (G-CSFR), Stat3α was constitutively activated in wild-type WT1-infected 32D cl3 in response to G-CSF, whereas, in WT1-uninfected 32D cl3, activation of Stat3α was only transient. However, most interesting was the fact that G-CSF stimulation resulted in constitutive activation of Stat3β only in wild-type WT1-infected 32D cl3, but not in WT1-uninfected 32D cl3. Thus, WT1 expression constitutively activated both Stat3α and Stat3β. A transient activation of Stat1 was detected in both wild-type WT1-infected and uninfected 32D cl3 after G-CSF stimulation, but no difference in its activation was found. No activation of MAP kinase was detected in both wild-type WT1-infected and uninfected 32D cl3 after G-CSF stimulation. These results demonstrated that WT1 expression competed with the differentiation-inducing signal mediated by G-CSFR and constitutively activated Stat3, resulting in the blocking of differentiation and subsequent proliferation. Therefore, the data presented here support our hypothesis that the WT1 gene plays an essential role in leukemogenesis and performs an oncogenic function in hematopoietic progenitor cells and represent the first demonstration of an important role of the WT1 gene in signal transduction in hematopoietic progenitor cells.
Naturally Occurring Mutations in Glycoprotein Ibα That Result in Defective Ligand Binding and Synthesis of a Truncated Protein
Authors: Kenny Dermot , Jónsson Ólafur G. , Morateck Patricia A. , Montgomery Robert R.
Published: October 2019

The platelet GPIb-V-IX complex is the receptor for the initial binding of von Willebrand factor (vWF) mediating platelet adhesion. The complex is composed of four membrane-spanning glycoproteins (GP): GPIbα, GPIbβ, GPIX, and GPV. Bernard-Soulier syndrome results from a qualitative or quantitative defect in one or more components of the platelet membrane GPIb-V-IX complex. We describe the molecular basis of a novel Bernard-Soulier syndrome variant in two siblings in whom GPIbα was not detected on the platelet surface but that was present in a soluble form in plasma. DNA sequence analysis showed that the affected individuals were compound heterozygotes for two mutations. One, inherited from a maternal allele, a T777 → C point mutation in GPIbα converting Cys65 → Arg within the second leucine rich repeat, the other, a single nucleotide substitution (G2078 → A) for the tryptophan codon (TGG) causing a nonsense codon (TGA) at residue 498 within the transmembrane region of GPIbα, inherited from a mutant paternal allele. The Bernard-Soulier phenotype was observed in siblings who were compound heterozygotes for these two mutations. Although GPIbα was not detected on the surface of the patient's platelets, soluble GPIbα could be immunoprecipitated from plasma. When plasmids encoding GPIbα containing the Cys65 → Arg mutation were transiently transfected into Chinese hamster ovary (CHO) cells stably expressing the GPβ-IX complex (CHOβIX), the expression of GPIbα was similar to the wild-type (WT) GPIbα, but did not bind vWF. When plasmids encoding GPIbα containing the Trp498 → stop were transiently transfected into CHOβIX, the surface expression of GPIbα was barely detectable compared with the WT GPIbα. Thus, this newly described compound heterozygous defect produces Bernard-Soulier syndrome by a combination of synthesis of a nonfunctional protein and of a truncated protein that fails to insert into the platelet membrane and is found circulating in plasma.
Expression of the CD8αβ-Heterodimer on CD8+ T Lymphocytes in Peripheral Blood Lymphocytes of Human Immunodeficiency Virus− and Human Immunodeficiency Virus+Individuals
Authors: Schmitz Jörn E. , Forman Meryl A. , Lifton Michelle A. , Concepción Orlando , Reimann Keith A. , Crumpacker Clyde S. , Daley John F. , Gelman Rebecca S. , Letvin Norman L.
Published: October 2019

CD8+ T lymphocytes play a pivotal role in controlling human immunodeficiency virus (HIV)-1 replication in vivo. We have performed four-color flow cytometric analysis of CD8+peripheral blood lymphocytes (PBL) from 21 HIV-1 seronegative and 103 seropositive individuals to explore the phenotypic heterogeneity of CD8β-chain expression on CD8+ T lymphocytes and to clarify how its expression on CD8+ T lymphocytes may relate to acquired immunodeficiency syndrome (AIDS) clinical progression. We showed that the single monoclonal antibody (MoAb) 2ST8-5H7, directed against the CD8αβ-heterodimer, identifies CD8+ T lymphocytes as effectively as the conventional combination of anti-CD3 and anti-CD8α antibodies. However, we detected a significantly lower mean fluorescence (MF) of anti-CD8αβ staining on PBL from HIV-1 seropositive donors as compared with seronegative donors. In fact, CD8+ T lymphocytes from HIV-1–infected individuals with the lowest CD4 counts showed the lowest levels of CD8αβ MF. To explore further this change in CD8αβ expression, we assessed the expression of 14 different cell surface molecules on CD8αβ+ T lymphocytes of PBL from 11 HIV-1 seronegative and 22 HIV-1 seropositive individuals. The MF of anti-CD8αβ staining was significantly reduced on CD8+T lymphocyte subsets that showed immunophenotypic evidence of activation. The subset of lymphocytes expressing low levels of CD8αβ expressed higher levels of activation, adhesion, and cytotoxic-associated molecules and was predominantly CD45RO+ and CD28−. Finally, we monitored the expression of the CD8αβ-heterodimer on PBL of eight HIV-1–infected individuals over a 16-week period after the initiation of highly active antiretroviral therapy (HAART), including zidovudine (ZDV), lamivudine (3TC), and indinavir (IDV), and found a significant increase in the expression of the CD8αβ-heterodimer. These results suggest that antibodies recognizing the CD8αβ-heterodimer are useful tools to specifically identify CD8+ T lymphocytes. Moreover, the quantitative monitoring of CD8αβ expression allows the detection of discrete CD8+ T lymphocyte subsets and may be useful for assessing the immune status of individuals infected with HIV-1.
Adhesion, Transendothelial Migration, and Reverse Transmigration of In Vitro Cultured Dendritic Cells
Authors: D'Amico Giovanna , Bianchi Giancarlo , Bernasconi Sergio , Bersani Laura , Piemonti Lorenzo , Sozzani Silvano , Mantovani Alberto , Allavena Paola
Published: October 2019

Dendritic cells (DC) are migratory cells which exhibit complex trafficking properties in vivo, involving interaction with vascular and lymphatic endothelium and extracellular matrix (ECM). The underlying mechanisms involved in these processes are still ill defined. In the present study we have investigated the ability of DC to interact in vitro with human vascular endothelial cells (EC) and ECM. DC were differentiated from monocytes by in vitro exposure to granulocyte-macrophage colony-stimulating factor and interleukin-13 for 7 days. In adhesion assays a considerable proportion of DC bound to resting EC monolayers: (17% ± 4%, mean ± SE of eight experiments). Adhesion to tumor necrosis factor (TNF)-activated EC was increased to 29% ± 5% (n = 8). Binding to resting EC was strongly inhibited by anti-CD11a and CD11b, but not by CD11c monoclonal antibodies (MoAbs); on TNF-activated EC, anti–VLA-4 in concert with anti-CD18 inhibited adhesion by more than 70%. Binding to a natural ECM, derived from cultured EC, or to purified fibronectin was high: 52% ± 6% (n = 8) involved VLA-4 and VLA-5 integrins. In a transmigration assay, 10% ± 2% (n = 6) of input cells were able to cross the EC monolayer. Unlike adhesion, transendothelial migration was significantly reduced by anti-CD31 MoAb. The amount of DC transmigrated through a monolayer of EC was increased twofold to threefold by a defined set of C-C chemokines including RANTES, MIP1α, MIP5, and, to a lesser extent, by MIP1β and MCP-3. Most importantly, in view of the trafficking pattern of these cells, a significant proportion of DC (13% ± 4% of input cells seeded) was able to migrate across the endothelial basement membrane and, subsequently, across the endothelial barrier (reverse transmigration). The adhesion molecules and chemoattractants characterized herein are likely to underlie the complex trafficking of DC in vivo.
Production of Interleukin-10 by Granulocyte Colony-Stimulating Factor–Mobilized Blood Products: A Mechanism for Monocyte-Mediated Suppression of T-Cell Proliferation
Authors: Mielcarek Marco , Graf Lynn , Johnson Gretchen , Torok-Storb Beverly
Published: October 2019

Previous reports showed that granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood mononuclear cells (G-PBMC) are hyporesponsive to alloantigen compared with control PBMC. In the current study, neutralizing antibodies to interleukin-10 (IL-10) increased the proliferative response of G-PBMC to alloantigen by 50.14% (± 12.79%; n = 8), whereas the proliferative response of control PBMC was not affected. The inhibition of OKT3-stimulated CD4 cell proliferation by G-PBMC–derived CD14+ cells could also be abrogated by the addition of IL-10 neutralizing antibodies. Further, IL-10 levels correlated with the number of CD14 cells in these cultures. Constitutive IL-10 mRNA levels detected by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) were 10-fold higher in G-PBMC compared with control PBMC. This translated into significantly higher IL-10 levels after 24-hour lipopolysaccharide (LPS) stimulation of G-PBMC compared with control PBMC (P = .036). IL-10 mRNA levels were also fivefold higher in isolated G-PBMC-derived CD14 cells compared with control CD14 cells. This corresponded to increased constitutive production of IL-10 by isolated G-PBMC–derived CD14 cells compared with control CD14 cells (357.2 ± 104.5 v 51.7 ± 30.5, P = .051). In conclusion, these data suggest that monocytes contained within G-PBMC, which, in comparison to marrow, are increased in absolute number and relative proportion to T cells, may suppress T-cell responsiveness by secretion of IL-10.
The Evolution of B Precursor Leukemia in the Eμ-ret Mouse
Authors: Wasserman Robert , Zeng Xiang-Xing , Hardy Richard R.
Published: October 2019

Eμ-ret mice carrying an RFP/RET fusion gene under the transcriptional control of the immunoglobulin heavy chain enhancer develop B lineage leukemias/lymphomas. We have characterized B-cell development in these mice before the onset of clinical disease to determine the steps involved in leukemogenesis. Flow cytometry reveals that the CD45R+CD43+CD24+BP-1+late pro–B-cell population is markedly expanded in the bone marrow of 3- to 5-week-old Eμ-ret mice. Compared with late pro–B cells from transgene-negative mice, Eμ-ret late pro–B cells have a limited capacity to differentiate in interleukin (IL)-7 and a higher incidence of VDJ rearrangements, but a similar cell cycle profile. In contrast, CD45R+CD43+CD24+BP-1−early pro–B cells from 3- to 5-week-old Eμ-ret mice, which also express the RFP/RET transgene, differentiate in IL-7 similarly to their normal counterparts. Furthermore, early pro–B cells from Eμ-ret and transgene-negative mice have an identical pattern of growth inhibition when exposed to interferons (IFNs)-α/β and -γ, whereas, pro–B-cell leukemia lines derived from Eμ-ret mice are markedly less sensitive to growth inhibition by these IFNs. In 13-week-old well-appearing Eμ-ret mice, late pro–B cells upregulate CYCLIN D1 expression and downregulate CASPASE-1 expression in a pattern that correlates with the emergence of B precursor cells in the peripheral blood and the loss of other B lineage subsets in the bone marrow. Taken together, these results suggest that the expression of the RFP/RET transgene initially prevents the normal elimination of late pro–B cells with nonproductive rearrangements. Secondary events that simultaneously disturb the normal transcriptional regulation of genes involved in the control of the cell cycle and apoptosis may allow for subsequent malignant transformation within the expanded late pro–B-cell population.
Temporal Synthesis of Band 3 Oligomers During Terminal Maturation of Mouse Erythroblasts. Dimers and Tetramers Exist in the Membrane as Preformed Stable Species
Authors: Hanspal Manjit , Golan David E. , Smockova Yva , Yi Scott J. , Cho Michael R. , Liu Shih-Chun , Palek Jiri
Published: October 2019

Band 3, the anion transport protein of the erythrocyte membrane, exists in the membrane as a mixture of dimers (B3D) and tetramers (B3T). The dimers are not linked to the skeleton and constitute the free mobile band 3 fraction. The tetramers are linked to the skeleton by their interaction with ankyrin. In this report we have examined the temporal synthesis and assembly of band 3 oligomers into the plasma membrane during red cell maturation. The oligomeric state of newly synthesized band 3 in early and late erythroblasts was analyzed by size-exclusion high-pressure liquid chromatography of band 3 extracts derived by mild extraction of plasma membranes with the nonionic detergent C12E8 (octaethylene glycol n-dodecyl monoether). This analysis revealed that at the early erythroblast stage, the newly synthesized band 3 is present predominantly as tetramers, whereas at the late stages of erythroid maturation, it is present exclusively as dimers. To examine whether the dimers and tetramers exist in the membrane as preformed stable species or whether they are interconvertible, the fate of band 3 species synthesized during erythroblast maturation was examined by pulse-chase analysis. We showed that the newly synthesized band 3 dimers and tetramers are stable and that there is no interconversion between these species in erythroblast membranes. Pulse-chase analysis followed by cellular fractionation showed that, in early erythroblasts, the newly synthesized band 3 tetramers are initially present in the microsomal fraction and later incorporated stably into the plasma membrane fraction. In contrast, in late erythroblasts the newly synthesized band 3 dimers move rapidly to the plasma membrane fraction but then recycle between the plasma membrane and microsomal fractions. Fluorescence photobleaching recovery studies showed that significant fractions of B3T and B3D are laterally mobile in early and late erythroblast plasma membranes, respectively, suggesting that many B3T-ankyrin complexes are unattached to the membrane skeleton in early erythroblasts and that the membrane skeleton has yet to become tightly organized in late erythroblasts. We postulate that in early erythroblasts, band 3 tetramers are transported through microsomes and stably incorporated into the plasma membrane. However, when ankyrin synthesis is downregulated in late erythroblasts, it appears that B3D are rapidly transported to the plasma membrane but then recycled between the plasma membrane and microsomal compartments. These observations may suggest novel roles for membrane skeletal proteins in stabilizing integral membrane protein oligomers at the plasma membrane and in regulating the endocytosis of such proteins.
Response at Three Months Is a Good Predictive Factor for Newly Diagnosed Chronic Myeloid Leukemia Patients Treated by Recombinant Interferon-
Authors: Mahon F.X. , Fabères C. , Pueyo S. , Cony-Makhoul P. , Salmi R. , Boiron J.M. , Marit G. , Bilhou-Nabera C. , Carrère A. , Montastruc M. , Pigneux A. , Bernard Ph. , Reiffers J.
Published: October 2019

In a single institution, we have used recombinant interferon- (IFN-) to treat 116 newly diagnosed Philadelphia-positive (Ph+) chronic myeloid leukemia (CML) patients and analyzed the predictive factors for response and survival. The patients whose median age was 50.3 years (range, 9 to 70) were administered IFN- (5 million units/m2/d) subcutaneously. The IFN- dose was subsequently adjusted to maintain the white blood cell and platelet counts between 1.5 and 5 × 109/L, 50 and 100 × 109/L, respectively. At diagnosis, the Sokal score was used to classify the patients into three groups: low (n = 57), intermediate (n = 42), and high risk (n = 16). A complete hematological response (CHR) was achieved in 93 cases (80.2%). Of the 116 patients, 113 were available for cytogenetic evaluation. Fifty patients (43%) achieved a major cytogenetic response (MCR) (=65% marrow Ph− cells), 37 of them having a complete cytogenetic response (CCR). The estimated 5-year survival of the 116 patients was 68% ± 11% (95% confidence interval [CI]) with a median follow-up of 42 months (range, 3 to 114) and 85% ± 11% (95% CI) with a median follow-up of 30.9 (range, 3 to 111) when patients were censored at the time of transplantation. Event-free survival at 5 years (adding death and transplant as event) was 46% ± 11% (95% CI). Using proportional hazards regression to study time-dependent variables, we confirmed that the most significant factor associated with survival was the cytogenetic response (MCR or CCR) (P < .0001). This factor was independent compared with the Sokal score and baseline variables used to calculate the Sokal score. Moreover, using either univariate or multivariate analysis, the achievement of CHR within 3 months was strongly correlated with MCR (P < .0001). Minimum cytogenetic response (mCR, ie, at least 5% of Ph− metaphases) at 3 months was also a significant predictive factor for MCR (P < .0001). These results show that IFN- can induce a high rate of hematological and cytogenetic response when administered in doses leading to myelosuppression. Factors such as the achievement of CHR and mCR within 3 months could be useful to identify early those patients who will not respond to IFN- and who need alternative treatments such as stem cell transplantation.
NF-κB Activation Is Required for C5a-Induced Interleukin-8 Gene Expression in Mononuclear Cells
Authors: Hsu Matthew H. , Wang Meiying , Browning Darren D. , Mukaida Naofumi , Ye Richard D.
Published: October 2019

C5a, a potent peptide chemoattractant, stimulates interleukin-8 (IL-8) secretion from peripheral blood mononuclear cells (PBMC). Experiments were conducted to understand the mechanisms for C5a-induced IL-8 production, which was 14-fold greater than that in unstimulated cells by 2 hours. IL-8 secretion was accompanied by accumulation of IL-8 mRNA in the cytosol and by nuclear expression of a κB DNA binding activity within 30 minutes. AP-1 but not NF-IL-6 DNA binding activity was also detected in C5a-stimulated PBMC; however, its delayed expression (maximal at 4 hours) suggested a less important role in the rapid production of IL-8. The correlation between C5a-induced κB binding activity and IL-8 gene expression was examined in the RAW264.7 macrophage cells using reporter genes directed by the κB sequence from IκB and IL-8 promoter regions. C5a-induced reporter gene expression was abolished by introducing mutations into the κB sites and by coexpression of a dominant negative IκB construct resistant to agonist-induced phosphorylation. Pertussis toxin, which ADP-ribosylates the Gi proteins known to couple to the C5a receptor, produced minimal inhibition of C5a-induced IL-8 expression and had little effect on C5a-induced calcium mobilization in RAW264.7 cells. These results suggest that NF-κB activation is required for C5a-induced IL-8 gene expression and that this response is mediated primarily through a pertussis toxin-insensitive pathway.
The Effect of Recombinant Human Erythropoietin on Platelet Counts Is Strongly Modulated by the Adequacy of Iron Supply
Authors: Loo Martine , Beguin Yves
Published: October 2019

The effect of recombinant human erythropoietin (rHuEpo) on megakaryopoiesis remains controversial. Treatment with rHuEpo in renal failure patients has been associated with a slight elevation of platelet counts. In animal studies, high doses of rHuEpo produced an increase of platelet counts followed by a gradual return to normal after 7 to 15 days or even a substantial degree of thrombocytopenia. However, because iron deficiency is also known to be associated with thrombocytosis, (functional) iron deficiency during rHuEpo could be contributing to these observations. We investigated the impact of iron supply on changes in platelet counts induced by rHuEpo. Rats were either fed normal food (normal rats) or received 1% carbonyl iron for 2 weeks or 3 months, as well as during the experiment, to achieve iron supplementation or overload, respectively. Rats of all three categories then received daily intravenous injections of rHuEpo (10, 50, or 150 U) or normal saline (0 U) for 20 days. With 0 to 10 U rHuEpo, platelets remained stable. In normal rats receiving 50 to 150 U rHuEpo, platelets increased to 120% to 140% of baseline at 4 to 12 days to level off at 120% at 16 to 20 days. This response was less sustained in splenectomized animals. Iron-supplemented rats receiving 50 to 150 U rHuEpo also increased platelets initially, but the peak was at day 4, followed by a gradual return to baseline and even a moderate thrombocytopenia later on. Iron-overloaded rats receiving 50 to 150 U rHuEpo also had increased platelets at day 4, but the duration of platelet increase was shorter, and they experienced a more pronounced degree of thrombocytopenia in proportion to the dose of rHuEpo. Because the early elevation of platelets was of larger magnitude than hematocrit changes, it is unlikely that it could be accounted for by shrinkage of plasma volume. Because it was observed in all three iron conditions, there appears to be some direct positive effect of rHuEpo on platelet production. However, after this transient effect, expanded erythropoiesis appears to exert a negative impact upon platelet production. Secondary thrombocytopenia was not related to splenic pooling, and its very slow correction after cessation of rHuEpo therapy is not compatible with changes in platelet survival. Rather, it is consistent with stem cell competition between erythroid and megakaryocytic development. However, this secondary thrombocytopenia is masked by (functional) iron deficiency in rats not receiving an adequate iron supply from food or stores.
Characterization of Mast Cell-Committed Progenitors Present in Human Umbilical Cord Blood
Authors: Kempuraj Duraisamy , Saito Hirohisa , Kaneko Azusa , Fukagawa Kazumi , Nakayama Masaharu , Toru Hano , Tomikawa Morimitsu , Tachimoto Hiroshi , Ebisawa Motohiro , Akasawa Akira , Miyagi Toko , Kimura Hiromitsu , Nakajima Toshiharu , Tsuji Kohichiro , Nakahata Tatsutoshi
Published: October 2019

Human mast cells are derived from CD34+ hematopoietic cells present in cord blood, bone marrow, and peripheral blood. However, little is known about the properties of the CD34+ cells. We demonstrated here that mast cell progenitors that have distinct phenotypes from other hematopoietic cell types are present in cord blood by culturing single, sorted CD34+ cells in 96-well plates or unsorted cells in methylcellulose. The CD34+ mast cell-committed progenitors often expressed CD38 and often lacked HLA-DR, whereas CD34+ erythroid progenitors often expressed both CD38 and HLA-DR and CD34+ granulocyte-macrophage progenitors often had CD33 and sometimes expressed CD38. We then cultured single cord blood-derived CD34+CD38+ cells under conditions optimal for mast cells and three types of myeloid cells, ie, basophils, eosinophils, and macrophages. Of 1,200 CD34+CD38+ cells, we were able to detect 13 pure mast cell colonies and 52 pure colonies consisting of either one of these three myeloid cell types. We found 17 colonies consisting of two of the three myeloid cell types, whereas only one colony consisted of mast cells and another cell type. These results indicate that human mast cells develop from progenitors that have unique phenotypes and that committed mast cell progenitors develop from multipotent hematopoietic cells through a pathway distinct from myeloid lineages including basophils, which have many similarities to mast cells.
Unicellular-Unilineage Erythropoietic Cultures: Molecular Analysis of Regulatory Gene Expression at Sibling Cell Level
Authors: Ziegler Benedikt L. , Müller Robert , Valtieri Mauro , Lamping Christa P. , Thomas Christian A. , Gabbianelli Marco , Giesert Christina , Bühring Hans-Jörg , Kanz Lothar , Peschle Cesare
Published: October 2019

In vitro studies on hematopoietic control mechanisms have been hampered by the heterogeneity of the analyzed cell populations, ie, lack of lineage specificity and developmental stage homogeneity of progenitor/precursor cells growing in culture. We developed unicellular culture systems for unilineage differentiation of purified hematopoietic progenitor cells followed by daughter cell analysis at cellular and molecular level. In the culture system reported here, (1) the growth factor (GF) stimulus induces cord blood (CB) progenitor cells to proliferate and differentiate/mature exclusively along the erythroid lineage; (2) this erythropoietic wave is characterized by less than 4% apoptotic cells; (3) asymmetric divisions are virtually absent, ie, nonresponsive hematopoietic progenitors with no erythropoietic potential are forced into apoptosis; (4) the system is cell division controlled (cdc), ie, the number of divisions performed by each cell is monitored. Single-cell reverse transcriptase-polymerase chain reaction (RT-PCR) analysis was applied to this culture system to investigate gene expression of diverse receptors, markers of differentiation, and transcription factors (EKLF, GATA-1, GATA-2, p45 NF-E2, PU.1, and SCL/Tal1) at discrete stages of erythropoietic development. Freshly isolated CD34+ cells expressed CD34, c-kit, PU.1, and GATA-2 but did not express CD36, erythropoietin receptor (EpoR), SCL/Tal1, EKLF, NF-E2, GATA-1, or glyocophorin A (GPA). In early to intermediate stages of erythroid differentiation we monitored the induction of CD36, Tal1, EKLF, NF-E2, and GATA-1 that preceeded expression of EpoR. In late stages of erythroid maturation, GPA was upregulated, whereas CD34, c-kit, PU.1, and GATA-2 were barely or not detected. In addition, competitive single-cell RT-PCR was used to assay CD34 mRNA transcripts in sibling CD34+CD38− cells differentiating in unilineage erythroid cultures: this analysis allowed us to semiquantitate the gradual downmodulation of CD34 mRNA from progenitor cells through their differentiating erythroid progeny. It is concluded that this novel culture system, coupled with single-cell RT-PCR analysis, may eliminate the ambiguities intrinsic to molecular studies on heterogeneous populations of hematopoietic progenitors/precursors growing in culture, particularly in the initial stages of development.
Ongoing Prothrombotic State in Patients With Antiphospholipid Antibodies: A Role for Increased Lipid Peroxidation
Authors: Praticò Domenico , Ferro Domenico , Iuliano Luigi , Rokach Joshua , Conti Fabrizio , Valesini Guido , FitzGerald Garret A. , Violi Francesco
Published: October 2019

We measured the urinary excretion of Isoprostane F2-III and Isoprostane-F2-VI, two markers of in vivo lipid peroxidation, and the circulating levels of the prothrombin fragment F1+2, a marker of thrombin generation, in 18 antiphospholipid antibodies-positive patients, in 18 antiphospholipid antibodies-negative patients with systemic lupus erythematosus, and in 20 healthy subjects. Furthermore, 12 patients positive for antiphospholipid antibodies were treated with (n = 7) or without (n = 5) antioxidant vitamins (vitamin E at 900 IU/d and vitamin C at 2,000 mg/d) for 4 weeks. Compared with antiphospholipid antibodies-negative patients, antiphospholipid antibodies-positive patients had higher urinary values of Isoprostane-F2-III (P = .0001), Isoprostane-F2-VI (P = .006), and plasma levels of the prothrombin fragment F1+2 (P= .0001). In antiphospholipid-positive patients, F1+2 significantly correlated with Isoprostane-F2-III (Rho = .56,P = .017) and Isoprostane-F2-VI (Rho = .61, P = .008). After 4 weeks of supplementation with antioxidant vitamins, we found a significant decrease in F1+2 levels (P < .005) concomitantly with a significant reduction of both Isoprostane-F2-III (P = .007) and Isoprostane-F2-VI (P < .005). No change of these variables was observed in patients not receiving antioxidant treatment. This study suggests that lipid peroxidation might contribute to the activation of clotting system in patients positive for antiphospholipid antibodies.
Agonist-Induced Regulation of Myosin Phosphatase Activity in Human Platelets Through Activation of Rho-Kinase
Authors: Suzuki Yoshinori , Yamamoto Masatoshi , Wada Hideo , Ito Masaaki , Nakano Takeshi , Sasaki Yasuharu , Narumiya Shuh , Shiku Hiroshi , Nishikawa Masakatsu
Published: October 2019

Human platelets contained about 15 times lower amounts of Rho-kinase than Ca2+/calmodulin-dependent myosin light chain (MLC) kinase. Anti–myosin-binding subunit (MBS) antibody coimmunoprecipitated Rho-kinase of human platelets, and addition of GTPγS-RhoA stimulated phosphorylation of the 130-kD MBS of myosin phosphatase and consequently inactivated myosin phosphatase. Two kinds of selective Rho-kinase inhibitors, HA1077 and Y-27632, reduced both GTPγS-RhoA–dependent MBS phosphorylation and inactivation of the phosphatase activity. Activation of human platelets with thrombin, a stable thromboxane A2 analog STA2, epinephrine, and serotonin resulted in an increase in MBS phosphorylation, and the agonist-induced MBS phosphorylation was prevented by pretreatment with the respective receptor antagonist. HA1077 and Y-27632 inhibited MBS phosphorylation in platelets stimulated with these agonists. These compounds also blocked agonist-induced inactivation of myosin phosphatase in intact platelets. In addition, HA1077 and Y-27632 inhibited 20-kD MLC phosphorylation at Ser19 and ATP secretion of platelets stimulated with STA2, thrombin (0.05 U/mL), and simultaneous addition of serotonin and epinephrine, whereas these compounds did not affect MLC phosphorylation or ATP secretion when platelets were stimulated with more than 0.1 U/mL thrombin. Thus, activation of Rho-kinase and the resultant phosphorylation of MBS is likely to be the common pathway for platelet activation induced by various agonists. These results also suggest that Rho-kinase–mediated MLC phosphorylation contributes to a greater extent to the platelet secretion induced by relatively weak agonists.
Vascular Endothelial Genes That Are Responsive to Tumor Necrosis Factor- In Vitro Are Expressed in Atherosclerotic Lesions, Including Inhibitor of Apoptosis Protein-1, Stannin, and Two Novel Genes
Authors: Horrevoets Anton J.G. , Fontijn Ruud D. , van Zonneveld Anton Jan , de Vries Carlie J.M. , ten Cate Jan Wouter , Pannekoek Hans
Published: October 2019

Activation and dysfunction of endothelial cells play a prominent role in patho-physiological processes such as atherosclerosis. We describe the identification by differential display of 106 cytokine-responsive gene fragments from endothelial cells, activated by monocyte conditioned medium or tumor necrosis factor-. A minority of the fragments (22/106) represent known genes involved in various processes, including leukocyte trafficking, vesicular transport, cell cycle control, apoptosis, and cellular protection against oxidative stress. Full-length cDNA clones were obtained for five novel transcripts that were induced or repressed more than 10-fold in vitro. These novel human cDNAs CA2_1, CG12_1, GG10_2, AG8_1, and GG2_1 encode inhibitor of apoptosis protein-1 (hIAP-1), homologues of apolipoprotein-L, mouse rabkinesin-6, rat stannin, and a novel 188 amino acid protein, respectively. Expression of 4 novel transcripts is shown by in situ hybridization on healthy and atherosclerotic vascular tissue, using monocyte chemotactic protein-1 as a marker for inflammation. CA2_1 (hIAP-1) and AG8_1 are expressed by endothelial cells and macrophage foam cells of the inflamed vascular wall. CG12_1 (apolipoprotein-L like) was specifically expressed in endothelial cells lining the normal and atherosclerotic iliac artery and aorta. These results substantiate the complex change in the gene expression pattern of vascular endothelial cells, which accompanies the inflammatory reaction of atherosclerotic lesions.
The T-Cell Activation Markers CD30 and OX40/CD134 Are Expressed in Nonoverlapping Subsets of Peripheral T-Cell Lymphoma
Authors: Jones Dan , Fletcher Christopher D.M. , Pulford Karen , Shahsafaei Aliakbar , Dorfman David M.
Published: October 2019

The tumor necrosis factor (TNF) receptor family includes several important markers of activation in T cells. We examined expression patterns of two T-cell-associated members of these receptors, namely CD30 and OX40/CD134, in 148 cases of T-cell lymphoma to identify possible objective immunohistochemical criteria for subclassification of these tumors. CD30 expression was characteristic of tumors with an anaplastic (46/47 cases [98%]) or large-cell (10/21 [48%]) morphology and was seen in only scattered cells in other tumor types. In contrast, large numbers of OX40/CD134+ tumors cells were typical of angioimmunoblastic lymphoma (15/16 [94%]), angiocentric lymphoma (4/4), a subset of large-cell lymphomas (10/21 [48%]), and lymphomas with a prominent histiocytic component (6/7 [86%]). Strong OX40/CD134 and CD30 coexpression was seen in only 4% of tumors, typically those with an anaplastic/Hodgkin’s-like appearance. OX40/CD134 expression was characteristic of tumors composed of activated CD4+ T cells and was not seen in small-cell T-cell lymphomas, lymphoblastic lymphomas, or other tumor types, including B-cell lymphomas or carcinomas. These results suggest that immunostaining for OX40/CD134 may be helpful in subclassification of peripheral T-cell lymphomas and that the patterns of TNF receptor family expression in these tumors may parallel those seen within nonneoplastic helper T-cell subsets.
Role of NF-κB in the Rescue of Multiple Myeloma Cells From Glucocorticoid-Induced Apoptosis by Bcl-2
Authors: Feinman Rena , Koury Jadd , Thames Michael , Barlogie Bart , Epstein Joshua , Siegel David S.
Published: October 2019

The molecular mechanisms by which multiple myeloma (MM) cells evade glucocorticoid-induced apoptosis have not been delineated. Using a human IgAκ MM cell line (ARP-1), we found that dexamethasone (Dex)-induced apoptosis is associated with decreased NF-κB DNA binding and κB-dependent transcription. Both nuclear p50:p50 and p50:p65 NF-κB complexes are detected in ARP-1 cells by supershift electrophoretic mobility shift assay (EMSA). Dex-mediated inhibition of NF-κB DNA binding precedes a notable increase in annexin V binding, thereby indicating that diminished NF-κB activity is an early event in Dex-induced apoptosis. Overexpression of bcl-2 in ARP-1 cells prevents Dex-mediated repression of NF-κB activity and apoptosis. Sustained NF-κB DNA binding is also observed in two previously characterized Dex-resistant MM cell lines (RPMI8226 and ARH-77) that express moderate levels of endogenous bcl-2 and IκB proteins. In addition, enforced bcl-2 expression in ARP-1 cells did not prevent the augmentation of IκB protein by Dex. We also noted a possible association between Dex-mediated downregulation of NF-κB in freshly obtained primary myeloma cells and the patients’ responsiveness to glucocorticoid-based chemotherapy. Collectively, our data suggest that the protective effects of bcl-2 in MM cells act upstream in the NF-κB activation–signaling pathway and the potential use of NF-κB as a biomarker in progressive MM.
Expression of Cell-Homologous Genes of Human Herpesvirus-8 in Human Immunodeficiency Virus-Negative Lymphoproliferative Diseases
Authors: Luppi Mario , Barozzi Patrizia , Maiorana Antonio , Trovato Raffaella , Marasca Roberto , Morselli Monica , Cagossi Katia , Torelli Giuseppe
Published: October 2019

Human herpesvirus-8 (HHV-8) genome encodes for genes homologous to human cellular genes such as interleukin-6 (IL-6), Cyclin-D, BCL-2, and IL-8 receptor (G-protein–coupled receptor [GCR]). We used reverse transcriptase-polymerase chain reaction to study the expression of these viral genes in lymphoproliferative disorders associated with HHV-8 infection. None of these genes was expressed in 1 case of benign, localized Castleman’s disease (CD), and only viral IL-6 and viral Cyclin-D were transcribed in 2 cases of benign lymphadenopathies with giant germinal center hyperplasia and increased vascularity. In contrast, all 4 genes were transcribed in 1 case of multicentric CD of plasma cell type with aggressive clinical course and in 1 primary effusion lymphoma cell line. Our study provides the evidence that various HHV-8 genes, homologous to cellular genes involved in control of proliferation and apoptosis, may be differently expressed in different lymphoid disorders in vivo.
Serum syndecan-1: a new independent prognostic marker in multiple myeloma
Authors: Seidel Carina , Sundan Anders , Hjorth Martin , Turesson Ingemar , Dahl Inger Marie S. , Abildgaard Niels , Waage Anders , Børset Magne
Published: October 2019

Serum samples drawn at diagnosis from 174 myeloma patients were analyzed for the presence of the heparin sulfate proteoglycan, syndecan-1. Syndecan-1 was elevated in 79% of patients (median, 643 units/mL) compared with 40 healthy controls (median, 128 units/mL),P < .0001. Serum syndecan-1 correlated with the following: serum creatinine, secretion of urine M-component over the course of 24 hours, soluble interleukin-6 (IL-6) receptor, C-terminal telopeptide of type I collagen, β2-microglobulin, percentage of plasma cells in the bone marrow, disease stage, and serum M-component concentration. In order to evaluate syndecan-1 as a prognostic marker in multiple myeloma, it was entered into a multivariate Cox regression model. Data from 138 patients were available for this analysis. As a continuous variable, syndecan-1 was an independent prognostic parameter in addition to serum β2-microglobulin and World Health Organization performance status. When syndecan-1 was dichotomized by the best cutoff (66th percentile, 1170 units/mL), the survival difference between the groups was highly significant: “high” syndecan-1 group had a median survival of 20 months, and the “low” syndecan-1 group had a median of 44 months (P < .0001). We conclude that syndecan-1 is a new independent prognostic parameter in multiple myeloma, and its role in prognostic classification systems should be further investigated.
CD13/N-aminopeptidase is involved in the development of dendritic cells and macrophages from cord blood CD34+ cells
Authors: Rosenzwajg Michelle , Tailleux Ludovic , Gluckman Jean Claude
Published: October 2019

Expression of CD13/N-aminopeptidase may reflect cell activation and growth. We examined its role regarding cell growth in cultures of cord blood CD34+ cells with stem cell factor/Flt-3 ligand/granulocyte-macrophage colony-stimulating factor/tumor necrosis factor-. Indeed, 82% ± 6% of cells from culture day 5 were CD13hi, 25% ± 8% of which were still Lin−. About 50% of CD13hiLin− cells, which comprise progenitors of dendritic cells (DC), monocytes/macrophages and granulocytes, and 30% of CD13loLin− cells were CD34+. Sorted CD34+CD13hiLin− cells, cultured further for 7 days with the same cytokines, expanded 31-fold and CD34-CD13hiLin− cells 7-fold, but CD34+CD13loLin− and CD34−CD13loLin− cells did not grow. Thus, cell growth correlated with CD13 expression, all the more so that cells were CD34+. Actinonin, the most potent N-aminopeptidase inhibitor, was used to engage CD13 on sorted CD13hiLin− cells and on culture day-7 bulk cells. In both cases, this resulted in reversible cell growth arrest, with 30% to 60% fewer cells in the G2/S-M phase than in controls. Interestingly, similar effects were noted with CD13 monoclonal antibody TÜK1, which does not inhibit N-aminopeptidase activity, but not with N-aminopeptidase-blocking antibodies WM15 and F23. All cycling cells appeared susceptible to actinonin, which induced cell apoptosis at the same time as Bcl-2 was downregulated and caspase-3 activity increased, but finally percentages and yields of DC and macrophage precursors were affected more than those of granulocytic cells. Thus, through engagement of N-aminopeptidase enzymatic site but possibly also of an independent determinant, CD13 plays a role in the growth of DC/macrophage progenitors and precursors.