bluuebunny/crossref_metadata_2025 huggingface dataset:
Mechanism of Interleukin-10 Inhibition of T-Helper Cell Activation by Superantigen at the Level of the Cell Cycle
Authors: Perrin George Q. , Johnson Howard M. , Subramaniam Prem S.
Published: October 2019

We have analyzed the effects of interleukin-10 (IL-10) on the entry of quiescent CD4+ T cells into the cell cycle upon stimulation with the superantigen staphylococcal enterotoxin B (SEB). IL-10 arrested cells at G0/G1. IL-10 treatment prevented the downregulation of p27Kip1, an inhibitory protein that controls progression out of the G0 phase of the cell cycle. IL-10 also prevented the upregulation of the G1 cyclins D2 and D3, proteins necessary for entry and progression through the G1 phase of the cell cycle. Associated with the inhibition of the cell cycle, IL-10 suppressed SEB induction of interleukin-2 (IL-2). Addition of exogenous IL-2 to IL-10–treated cells significantly reversed the antiproliferative effects of IL-10. Moreover, IL-10 effects on the early G1proteins p27Kip1 and cyclin D2 were similarly reversed by exogenous IL-2. Although this reversal by IL-2 was pronounced, it was not complete, suggesting that IL-10 may have some effects not directly related to the suppression of IL-2 production. Cell separation experiments suggest that IL-10 can effect purified CD4+ T cells directly, providing functional evidence for the presence of IL-10 receptors on CD4+ T cells. IL-10 also inhibited expression of IL-2 transcriptional regulators c-fos and c-jun, which also inhibit other cell functions. Our studies show that the mechanism of IL-10 regulation of quiescent CD4+ T-cell activation is mainly by blocking induction of IL-2 that is critical to downregulation of p27Kip1 and upregulation of D cyclins in T-cell activation and entry into the cell cycle.
Insulin-Like Growth Factor-1 Acts as a Chemoattractant Factor for 5T2 Multiple Myeloma Cells
Authors: Vanderkerken Karin , Asosingh Kewal , Braet Filip , Van Riet Ivan , Van Camp Ben
Published: October 2019

The chemotactic and growth-stimulatory effect of insulin-like growth factor 1 (IGF-1) was investigated in the experimental mouse 5T2 multiple myeloma (MM) model. Chemotaxis was analyzed by classical checkerboard analysis. Bone marrow fibroblasts–conditioned medium exhibited a chemotactic effect on 5T2 MM cells that could be neutralized by adding a blocking antibody to IGF-1. On the other hand, exogenously added IGF-1 also had a chemotactic effect on the 5T2 MM cells. Moreover, in vitro analysis demonstrated that transmigrated 5T2 MM cells have a higher expression of IGF-1 receptor, both in bone marrow–conditioned medium and in IGF-1–induced chemotaxis, in comparison to cells before migration. When analyzed in vivo, 18 hours after injection of the heterogeneous 5T2 MM population, 5T2 MM cells present in the bone marrow show a higher expression of the IGF-1 receptor than their counterparts before injection. When the proliferative effect of IGF-1 was analyzed, no stimulation was observed, which is in contrast to the influence of bone marrow–conditioned medium and interleukin-6. Our results suggest a causal relationship between the presence of IGF-1 in the bone marrow and the chemotaxis of MM cells to and their subsequent presence in the bone marrow.
Hairy Cell Leukemia-Specific Recognition by Multiple Autologous HLA-DQ or DP-Restricted T-Cell Clones
Authors: van de Corput Lisette , Kluin-Nelemans Hanneke C. , Kester Michel G.D. , Willemze Roel , Falkenburg J.H. Frederik
Published: October 2019

We studied in patients with hairy cell leukemia (HCL) whether autoreactive T cells could be isolated with specific reactivity to the HCL cells. HCL cells were activated via triggering of CD40 on the cell membrane and used as stimulator cells to generate autologous T-cell clones. Two types of CD4+BV2+ T-cell clones with different CDR3 rearrangements and one type of CD4+BV8S3+ T-cell clone were generated from the spleen or blood. These clones specifically recognized the autologous HCL cells, without reactivity to autologous peripheral blood mononuclear cells (PBMC), phytohemagglutinin blasts, or Epstein-Barr virus–transformed B cells in a primed lymphocyte test. Blocking and panel studies using HCL cells from 11 other patients showed that recognition of the HCL cells by the BV2+ T cells was restricted by HLA-DQA1*03/DQB1*0301, and the BV8S3+ T cells were restricted by DPB1*04. The T-cell clones did not recognize DPB1*04+ or DQ3+ PBMC from healthy donors or DP/DQ matched malignant cells from patients with other hematologic malignancies, except for one patient with acute lymphoblastic leukemia. These HCL-specific T-cell clones may be used for the detection of an HCL-specific tumor antigen.
BCR/ABL− CD34+HLA-DR−Progenitor Cells in Early Chronic Phase, But Not in More Advanced Phases, of Chronic Myelogenous Leukemia Are Polyclonal
Authors: Delforge Michel , Boogaerts Marc A. , McGlave Philip B. , Verfaillie Catherine M.
Published: October 2019

Chronic myelogenous leukemia (CML) is characterized by the Philadelphia (Ph) translocation and BCR/ABL gene rearrangement which occur in a pluripotent hematopoietic progenitor cell. Ph-negative (Ph−) hematopoiesis can be restored in vivo after treatment with -interferon or intensive chemotherapy, suggesting that normal stem and progenitor cells coexist with the Ph+ clone. We have previously shown that Ph− progenitors are highly enriched in the CD34+HLA-DR− fraction from early chronic phase (ECP) CML patients. Previous studies have suggested that the Ph-translocation represents a secondary clonal hit occurring in an already clonally mutated Ph− progenitor or stem cells, leaving the unanswered question whether Ph−CD34+HLA-DR- progenitors are normal. To show the clonal nature of Ph−CD34+HLA-DR− CML progenitors, we have compared the expression of BCR/ABL mRNA with X-chromosome inactivation patterns (HUMARA) in mononuclear cells and in CD34+HLA-DR+ and CD34+HLA-DR− progenitors in marrow and blood obtained from 11 female CML patients (8 in chronic phase and 3 in accelerated phase [AP] disease). Steady-state marrow-derived BCR/ABL mRNA−, CD34+HLA-DR−progenitors had polyclonal X-chromosome inactivation patterns in 2 of 2 patients. The same polyclonal pattern was found in the progeny of CD34+HLA-DR− derived long-term culture-initiating cells. Mobilization with intensive chemotherapy induced a Ph−, BCR/ABL mRNA−and polyclonal state in the CD34+HLA-DR−and CD34+HLA-DR+ progenitors from 2 ECP patients. In a third ECP patient, polyclonal CD34+ cells could only be found in the first peripheral blood collection. In contrast to ECP CML, steady-state marrow progenitors in late chronic phase and AP disease were mostly Ph+, BCR/ABL mRNA+, and clonal. Further, in the majority of these patients, a Ph−, polyclonal state could not be restored despite mobilization with intensive chemotherapy. We conclude from these studies that CD34+HLA-DR− cells that are Ph− and BCR/ABL mRNA− are polyclonal and therefore benign. This population is suitable for autografting in CML.
Nitric Oxide–Mediated Augmentation of Polymorphonuclear Free Radical Generation After Hypoxia-Reoxygenation
Authors: Sethi Sonia , Singh Mahendra Pratap , Dikshit Madhu
Published: October 2019

Polymorphonuclear leukocytes (PMNLs), nitric oxide (NO), calcium, and free radicals play an important role in hypoxia/ischemia and reoxygenation injury. In the present study, NO donors, sodium nitroprusside (SNP), and diethylamine-NO (DEA-NO) at low concentrations (10 and 100 nmol/L) potentiated, while higher (10 μmol/L to 10 mmol/L) concentrations inhibited free radical generation response in the rat PMNLs. Free radical generation response was found to be significantly augmented when hypoxic PMNLs were reoxygenated (hypoxia-reoxygenation [H-R]). This increase in free radical generation after reoxygenation or SNP (10 nmol/L) was blocked in the absence of extracellular calcium. SNP (10 nmol/L) or H-R–mediated increases in the free radical generation were prevented by the pretreatment of PMNLs with NO scavenger (hemoglobin), the polyadenine diphosphate (ADP)-ribosylation synthase inhibitor (benzamide) or the calcium channel antagonist (felodipine). A significant augmentation in the nitrite and intracellular calcium levels was observed during hypoxia. Hemoglobin pretreatment also blocked the increase in intracellular calcium levels due to SNP (10 nmol/L) or hypoxia. Thus, increased availability of NO during SNP treatment or H-R, may have led to an ADP-ribosylation–mediated increase in intracellular calcium, thereby increasing the free radical generation from the rat PMNLs.
Peripheral Blood CD14+ Cells From Healthy Subjects Carry a Circular Conformation of Latent Cytomegalovirus Genome
Authors: Bolovan-Fritts Cynthia A. , Mocarski Edward S. , Wiedeman Jean A.
Published: October 2019

The majority of the human population harbors latent cytomegalovirus. Although CD14+ peripheral blood mononuclear cells have been implicated as sites of latency, the conformation of the latent viral genome in these cells is unknown. In this study, the conformation of viral genomic DNA was assessed in CD14+ cells from healthy virus seropositive carriers using an electrophoretic separation on native agarose gels in combination with polymerase chain reaction detection. Here we show that the viral genome migrates as a circular plasmid with a mobility equivalent to a circular 230-kb Shigella flexneri megaplasmid marker. Neither linear nor complex or integrated forms of the viral genome were detected. This report provides further evidence that the CD14+ cell population is an important site of viral latency in the naturally infected human host. Detection of the viral genome as a circular plasmid during latency suggests that this virus maintains its genome in a manner analogous to other herpesviruses where latent viral genome conformation has been studied.
Murine T Lymphocytes Incapable of Producing Macrophage Inhibitory Protein-1 Are Impaired in Causing Graft-Versus-Host Disease Across a Class I But Not Class II Major Histocompatibility Complex Barrier
Authors: Serody Jonathan S. , Cook Donald N. , Kirby Suzanne L. , Reap Elizabeth , Shea Thomas C. , Frelinger Jeffrey A.
Published: October 2019

The routine use of bone marrow transplantation is limited by the occurrence of acute and chronic graft-versus-host disease (GVHD). Current approaches to decreasing the occurrence of GVHD after allogeneic transplantation use T-cell depletion, use immunosuppressive agents, or block costimulatory molecule function. The role of proteins in the recruitment of alloreactive lymphocytes has not been well characterized. Chemokines are a large family of proteins that mediate recruitment of mononuclear cells in vitro and in vivo. To investigate the role of T-cell production of the chemokine macrophage inhibitory protein-1 (MIP-1) in the occurrence of GVHD, splenocytes either from wild-type or from MIP-1−/− mice were administered to class I (B6.C-H2bm1) and class II disparate mice (B6-C-H2bm12). The incidence and severity of GVHD was markedly reduced in bm1 mice receiving splenocytes from MIP-1−/− mice as compared with mice receiving wild-type splenocytes. Bm1 mice receiving MIP-1−/− splenocytes had significantly less weight loss and markedly reduced inflammatory responses in the lung and liver than mice receiving C57BL/6 splenocytes. Bm1 mice receiving MIP-1−/− splenocytes had a markedly decreased production of antichromatin autoantibodies and impaired generation of bm1-specific T lymphocytes versus wild-type mice. However, MIP-1−/− splenocytes easily induced GVHD when administered to bm12 mice. This data show that blockade of chemokine production or function may provide a new approach to the prevention or treatment of GVHD but that chemokines that recruit both CD4+ and CD8+ lymphocytes may need to be targeted.
Definitive But Not Primitive Hematopoiesis Is Impaired injumonji Mutant Mice
Authors: Kitajima Kenji , Kojima Mizuyo , Nakajima Kuniko , Kondo Shunzo , Hara Takahiko , Miyajima Atsushi , Takeuchi Takashi
Published: October 2019

A novel gene, jumonji was identified by a mouse gene trap strategy. The jumonji gene encodes a protein containing a putative DNA binding domain. The mice homozygous for jumonjigene with a BALB/cA genetic background show hypoplasia of the fetal liver and embryonic lethality, suggesting impaired hematopoiesis. In the peripheral blood of jumonji mutant embryos, the number of fetal liver–derived definitive erythrocytes, but not yolk sac–derived primitive erythrocytes, showed a marked reduction, suggesting thatjumonji mutants die of anemia. The defects of definitive erythrocytes in jumonji mutants seemed to be caused by a decrease in the numbers of multiple hematopoietic progenitors including colony-forming unit-spleen (CFU-S) in the fetal liver. However, hematopoietic stem cells (HSCs) in the fetal liver of jumonjimutants could reconstitute the hematopoietic system of lethally irradiated recipients. In the fetal liver, the jumonji gene is expressed in fibroblastic cells and endothelial cells, but not in Lin−/c-Kit+/Sca-1+ cells known to include HSCs. These results suggest that an environmental defect induce the impaired hematopoiesis in the fetal liver ofjumonji mutant embryos.
Compound-Heterozygous Mutations in the Plasminogen Gene Predispose to the Development of Ligneous Conjunctivitis
Authors: Schuster Volker , Seidenspinner Silvia , Zeitler Petra , Escher Cornelia , Pleyer Uwe , Bernauer Wolfgang , Stiehm E. Richard , Isenberg Sherwin , Seregard Stefan , Olsson Thomas , Mingers Anne-Marie , Schambeck Christian , Kreth Hans Wolfgang
Published: October 2019

Homozygous type I plasminogen deficiency has been identified as a cause of ligneous conjunctivitis. In this study, 5 additional patients with ligneous conjunctivitis are examined. Three unrelated patients (1 boy, 1 elderly woman, and 1 man) had plasminogen antigen levels of less than 0.4, less than 0.4, and 2.4 mg/dL, respectively, but had plasminogen functional residual activity of 17%, 18%, and 17%, respectively. These subjects were compound-heterozygotes for different missense mutations in the plasminogen gene: Lys19 → Glu/Arg513 → His, Lys19 → Glu/Arg216 → His, and Lys19 → Glu/Leu128 → Pro, respectively. The other 2 patients, a 14-year-old boy and his 19-year-old sister, who both presented with a severe course of the disease, exhibited plasminogen antigen and functional activity levels below the detection limit (<0.4 mg/dL and <5%, respectively). These subjects were compound-heterozygotes for a deletion mutation (del Lys212) and a splice site mutation in intron Q (Ex17 + 1del-g) in the plasminogen gene. These findings show that certain compound-heterozygous mutations in the plasminogen gene may be associated with ligneous conjunctivitis. Our findings also suggest that the severity of clinical symptoms of ligneous conjunctivitis and its associated complications may depend on the amount of plasminogen functional residual activity.
Interleukin-4 Synergizes With Raf-1 to Promote Long-Term Proliferation and Activation of c-jun N-terminal Kinase
Authors: Levings Megan K. , Bessette Darrell C. , Schrader John W.
Published: October 2019

This report shows that interleukin-4 (IL-4), which plays a key role in regulating immune responses, fails to support cellular growth. We investigated whether this failure of IL-4 to promote growth was because of its unique inability to activate the Ras/Raf/Erk pathway. Consistent with other reports, expression in Ba/F3, a factor-dependent hematopoietic cell line, of either activated Q61KN-Ras or a hormone-inducible activated Raf-1, resulted in suppression of apoptosis but not in long-term growth. However, in the presence of IL-4, Ba/F3 cells that expressed either Q61KN-Ras or activated Raf-1 grew continuously at a rate comparable with that stimulated by IL-3. Investigation of the biochemical events associated with the stimulation of long-term growth showed that, as expected, the presence of activated Raf-1 resulted in an increased activity of extracellular signal regulated kinase (ERK) mitogen-activated protein kinase (MAPK) but not of c-jun N-terminal kinase/stress-activated protein kinase (JNK). However, surprisingly, if IL-4 was present, cells expressing active Raf-1 exhibited increases in JNK activity. These observations point to a novel mechanism for JNK activation involving synergy between Raf-1 and pathways activated by IL-4 and suggest that in hematopoietic cells proliferation is correlated not only with “mitogen activated” ERK activity, but also with JNK activity.
A Common Signaling Pathway Via Syk and Lyn Tyrosine Kinases Generated From Capping of the Sialomucins CD34 and CD43 in Immature Hematopoietic Cells
Authors: Tada Jun-ichi , Omine Mitsuhiro , Suda Toshio , Yamaguchi Naoto
Published: October 2019

The sialomucin CD34 is a useful marker for hematopoietic stem/progenitor cells. However, the role of CD34 remains poorly understood. Here we investigate the functions of CD34 and another sialomucin CD43 coexpressed on hematopoietic stem/progenitor cells. Stimulation of undifferentiated hematopoietic KG1a cells with anti-CD34 or anti-CD43 induced homotypic cytoadhesion, accompanied by formation of a long-lived cap of CD34 and CD43 respectively, which colocalized with F-actin. Stimulation with either antibody specifically increased tyrosine phosphorylation of the identical set of proteins of Lyn, Syk, pp60, pp69, and pp77 at the capping site. These events were similar to those observed in monocytic U937 cells ectopically expressing CD34. After stimulation of KG1a cells, coimmunoprecipitation of Lyn with pp69 and pp77 and of Syk with pp37 was detected in the membrane fraction. Blockade of antibody-induced cap formation by treatment with cytochalasin D leads to inhibition of tyrosine phosphorylation of Syk and pp77 and homotypic cytoadhesion. Moreover, normal human CD34+ bone marrow cells showed cap formation of CD34 or CD43 after stimulation. These results suggest that crosslinking of either CD34 or CD43 activates the same signaling pathway for cytoadhesion through Lyn, Syk, and the novel tyrosine-phosphorylated proteins within hematopoiesis.
Posttranslational Regulation of Myc Function in Response to Phorbol Ester/Interferon-γ–Induced Differentiation of v-Myc–Transformed U-937 Monoblasts
Authors: Bahram Fuad , Wu Siqin , Öberg Fredrik , Lüscher Bernhard , Larsson Lars-Gunnar
Published: October 2019

The transcription factors of the Myc/Max/Mad network are important regulators of cell growth, differentiation, and apoptosis and are frequently involved in tumor development. Constitutive expression of v-Myc blocks phorbol ester (TPA)-induced differentiation of human U-937 monoblasts. However, costimulation with interferon-γ (IFN-γ) and TPA restores terminal differentiation and G1cell-cycle arrest despite continuous expression of v-Myc. The mechanism by which TPA + IFN-γ counteract v-Myc activity has not been unravelled. Our results show that TPA + IFN-γ treatment led to an inhibition of v-Myc– and c-Myc–dependent transcription, and a specific reduction of v-Myc:Max complexes and associated DNA-binding activity, whereas the steady state level of the v-Myc protein was only marginally affected. In contrast, TPA + IFN-γ costimulation neither increased the expression of Mad1 or other mad/mnt family genes nor altered heterodimerization or DNA-binding activity of Mad1. The reduced amount of v-Myc:Max heterodimers in response to treatment was accompanied by partial dephosphorylation of v-Myc and c-Myc. Phosphatase treatment of Myc:Max complexes lead to their dissociation, thus mimicking the effect of TPA + IFN-γ. In addition to modulation of the expression of Myc/Max/Mad network proteins, posttranslational negative regulation of Myc by external signals may, therefore, be an alternative biologically important level of control with potential therapeutic relevance for hematopoietic and other tumors with deregulated Myc expression.
Humanized Anti-HM1.24 Antibody Mediates Myeloma Cell Cytotoxicity That Is Enhanced by Cytokine Stimulation of Effector Cells
Authors: Ozaki Shuji , Kosaka Masaaki , Wakahara Yuji , Ozaki Yasuko , Tsuchiya Masayuki , Koishihara Yasuo , Goto Tetsuya , Matsumoto Toshio
Published: October 2019

To develop a new immunotherapy for multiple myeloma, we have generated a monoclonal antibody (MoAb) that detects a human plasma cell-specific antigen, HM1.24. Our previous study has shown that mouse anti-HM1.24 MoAb inhibits the proliferation of human myeloma cells implanted into severe combined immunodeficiency mice. In this report, we evaluated the antitumor activity of the humanized anti-HM1.24 MoAb (IgG1κ), which was constructed by grafting the complementarity-determining regions. In contrast to the parent mouse MoAb, humanized anti-HM1.24 MoAb mediated antibody-dependent cellular cytotoxicity (ADCC) against both myeloma cell lines and myeloma cells from patients in the presence of human peripheral blood mononuclear cells (PBMCs). The PBMCs from untreated myeloma patients exhibited ADCC activity as efficiently as those of healthy donors. Although decreased ADCC activity of PBMCs was observed in patients who responded poorly to conventional chemotherapy, it could be significantly augmented by the stimulation with interleukin-2 (IL-2), IL-12, or IL-15. There was a strong correlation between the percentage of CD16+ cells and ADCC activity in the PBMCs of myeloma patients. Moreover, peripheral blood stem cell collections from myeloma patients contained higher numbers of CD16+ cells than PBMCs and exhibited ADCC activity that was enhanced by IL-2. These results indicate that humanized anti-HM1.24 MoAb has potential as a new therapeutic strategy in multiple myeloma and that treatment of effector cells with immunomodulating cytokines can restore the effect of humanized anti-HM1.24 MoAb in patients with diminished ADCC activity.
Alteration of Actin Organization by Jaspamide Inhibits Ruffling, but not Phagocytosis or Oxidative Burst, in HL-60 Cells and Human Monocytes
Authors: Fabian Ina , Halperin Drora , Lefter Smadar , Mittelman Leonid , Altstock Rom T. , Seaon Osnat , Tsarfaty Ilan
Published: October 2019

Jaspamide, a naturally occurring cyclic peptide isolated from the marine sponge Hemiastrella minor, has fungicidal and growth-inhibiting activities. Exposure of promyelocytic HL-60 cells and human monocytes to jaspamide induces a dramatic reorganization of actin from a typical fibrous network to focal aggregates. HL-60 cells exposed to 5 × 10−8 mol/L or 10−7 mol/L jaspamide exhibited a reduced proliferation rate. In addition, 10−7mol/L jaspamide induced maturation of HL-60 cells as indicated by the appearance of a lobulated nucleus in 55% ± 5% of the cells and immunophenotypic maturation of the leukemia cells (upregulation of CD16 and CD14 B antigens). Further characterization has shown that F-actin is aggregated both in HL-60 cells and in human monocytes exposed to 10−7 mol/L jaspamide. Well-spread cultured human monocytes contracted and adopted round shapes after treatment with jaspamide. Moreover, a dose-dependent increase in both total actin and de novo synthesized portions of the soluble actin was observed in jaspamide-treated HL-60 cells. Jaspamide treatment inhibits ruffling and intracellular movement in HL-60 cells and monocytes, but does not affect phagocytic activity or respiratory burst activity. The consequential effects of jaspamide-induced actin reorganization on ruffling, versus its negligible effect on phagocytosis and oxidative burst, may shed light on molecular mechanisms of actin involvement in these processes. Jaspamide disrupts the actin cytoskeleton of normal and malignant mammalian cells with no significant effect on phagocytic activity and may, therefore, be considered as a novel therapeutic agent.
Angiogenesis and Hematopoiesis Induced by Kaposi’s Sarcoma-Associated Herpesvirus-Encoded Interleukin-6
Authors: Aoki Yoshiyasu , Jaffe Elaine S. , Chang Yuan , Jones Karen , Teruya-Feldstein Julie , Moore Patrick S. , Tosato Giovanna
Published: October 2019

Kaposi’s sarcoma-associated herpesvirus (KSHV; also known as human herpesvirus 8 [HHV-8]) is a herpesvirus linked to the development of Kaposi’s sarcoma (KS), primary effusion lymphoma, and a proportion of Castleman’s disease. KSHV encodes viral interleukin-6 (vIL-6), which is structurally homologous to human and murine IL-6. The biological activities of vIL-6 are largely unknown. To gain insight into the biology of vIL-6, we expressed vIL-6 in murine fibroblasts NIH3T3 cells and inoculated stable vIL-6–producing clones into athymic mice. vIL-6 was detected selectively in the blood of mice injected with vIL-6–expressing clones. Compared with controls, vIL-6–positive mice displayed increased hematopoiesis in the myeloid, erythroid, and megakaryocytic lineages; plasmacytosis in spleen and lymph nodes; hepatosplenomegaly; and polyclonal hypergammaglobulinemia. vIL-6–expressing NIH3T3 cells gave rise to tumors more rapidly than did control cells, and vIL-6–positive tumors were more vascularized than controls. Vascular endothelial growth factor (VEGF) was detected at higher levels in the culture supernatant of vIL-6–expressing cells compared with controls, and immunohistochemical staining detected VEGF in spleen, lymph nodes, and tumor tissues from mice bearing vIL-6–producing tumors but not control tumors. Thus, vIL-6 is a multifunctional cytokine that promotes hematopoiesis, plasmacytosis, and angiogenesis. Through these functions, vIL-6 may play an important role in the pathogenesis of certain KSHV-associated disorders.
Altered Multidrug Resistance Phenotype Caused by Anthracycline Analogues and Cytosine Arabinoside in Myeloid Leukemia
Authors: Hu Xiu F. , Slater Alison , Kantharidis Phillip , Rischin Danny , Juneja Surender , Rossi Ralph , Lee Grace , Parkin John D. , Zalcberg John R.
Published: October 2019

The expression of P-glycoprotein (Pgp) is often increased in acute myeloid leukemia (AML). However, little is known of the regulation of Pgp expression by cytotoxics in AML. We examined whether Pgp expression and function in leukemic blasts was altered after a short exposure to cytotoxics. Blasts were isolated from 19 patients with AML (15 patients) or chronic myeloid leukemia in blastic transformation (BT-CML, 4 patients). Pgp expression and function were analyzed by flow cytometric analysis of MRK 16 binding and Rhodamine 123 retention, respectively. At equitoxic concentrations, ex vivo exposure for 16 hours to the anthracyclines epirubicin (EPI), daunomycin (DAU), idarubicin (IDA), or MX2 or the nucleoside analogue cytosine arabinoside (AraC) differentially upregulated MDR1/Pgp expression in Pgp-negative and Pgp-positive blast cells. In Pgp-negative blasts, all four anthracyclines and AraC significantly increased Pgp expression (P = .01) and Pgp function (P = .03). In contrast, MX2, DAU, and AraC were the most potent in inducing Pgp expression and function in Pgp positive blasts (P < .05). A good correlation between increased Pgp expression and function was observed in Pgp-negative (r = .90, P = .0001) and Pgp-positive blasts (r = .77,P = .0002). This increase in Pgp expression and function was inhibited by the addition of 1 μmol/L PSC 833 to blast cells at the time of their exposure to these cytotoxics. In 1 patient with AML, an increase in Pgp levels was observed in vivo at 4 and 16 hours after the administration of standard chemotherapy with DAU/AraC. Upregulation of Pgp expression was also demonstrated ex vivo in blasts harvested from this patient before the commencement of treatment. In 3 other cases (1 patient with AML and 2 with BT-CML) in which blasts were Pgp negative at the time of initial clinical presentation, serial samples at 1 to 5 months after chemotherapy showed the presence of Pgp-positive blasts. All 3 patients had refractory disease. Interestingly, in all 3 cases, upregulation of Pgp by cytotoxics was demonstrated ex vivo in blasts harvested at the time of presentation. These data suggest that upregulation of the MDR1 gene may represent a normal response of leukemic cells to cytotoxic stress and may contribute to clinical drug resistance.
Sequential Homoharringtonine and Interferon- in the Treatment of Early Chronic Phase Chronic Myelogenous Leukemia
Authors: O’Brien Susan , Kantarjian Hagop , Koller Charles , Feldman Eric , Beran Miloslav , Andreeff Michael , Giralt Sergio , Cheson Bruce , Keating Michael , Freireich Emil , Rios Mary Beth , Talpaz Moshe
Published: October 2019

Homoharringtonine (HHT) is a novel plant alkaloid that produced a complete hematologic remission (CHR) in 72% of patients with late chronic phase chronic myelogenous leukemia (CML). Cytogenetic (CG) remissions were noted in 31%. In this study, six courses of HHT were administered to 90 patients with early chronic phase CML (< 1 year from diagnosis). Patients then received interferon- (IFN-) with a target dose of 5 MU/m2 daily. Results were compared with those in a prior group of patients treated with IFN-–based therapy between 1982 and 1990. Ninety-two percent of patients achieved CHR with HHT; CG responses were observed in 60% and were major in 27%. Both CHR and CG response rates were significantly higher than those seen in historical control patients after 6 months of IFN- therapy. After receiving HHT, patients required lower doses of IFN- to maintain a CHR. The median dose delivered was 2.4 MU/m2. This reduction in IFN- dose was associated with a lower incidence of myalgia and gastrointestinal (GI) disturbances than that seen in patients treated at the 5 MU/m2 dose. Overall, CG responses were seen in 66% of the patients who received HHT and IFN- compared with 61% of the historical control patients. HHT is a very effective treatment of early chronic phase CML, and ongoing trials are investigating the simultaneous administration of HHT and IFN-, as well as that of HHT and low-dose cytosine arabinoside in patients failing IFN- therapy.
Some Anticardiolipin Antibodies Recognize a Combination of Phospholipids With Thrombin-Modified Antithrombin, Complement C4b-Binding Protein, and Lipopolysaccharide Binding Protein
Authors: Arvieux Josiane , Pernod Gilles , Regnault Véronique , Darnige Luc , Garin Jérôme
Published: October 2019

The standard enzyme-linked immunosorbent assay (ELISA) for anticardiolipin antibodies (ACA) detects a heterogenous group of antibodies against cardiolipin on its own, β2-glycoprotein I (β2GPI), and, potentially, other phospholipid-binding plasma proteins from bovine or human origin. In an attempt to identify new proteic targets of ACA, we selected 6 patients who possessed cofactor-dependent ACA but no antibody to human or bovine β2GPI detectable in the β2GPI-ELISA. Three of these samples proved to recognize β2GPI in combination with cardiolipin, but not β2GPI directly immobilized on γ-irradiated polystyrene or agarose beads. In the other cases, the component required for ACA binding was purified from adult bovine serum or plasma by means of ammonium sulfate precipitation and chromatography on Phenyl-Sepharose, diethyl aminoethyl (DEAE)-cellulose, heparin-Ultrogel, and Sephacryl S-300 columns. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis coupled to N-terminal amino acid microsequencing identified the cofactors of patients no. 4, 5, and 6 ACA as lipopolysaccharide binding protein (LBP), complement C4b-binding protein (C4BP), and the thrombin-antithrombin (AT) complex, respectively. Adsorption of each of these cofactor preparations with cardiolipin liposomes led to suppression of ACA reactivity, concomitant with the loss of bands from SDS gels corresponding to sequenced material. Bacterial lipopolysaccharide (which forms high-affinity complexes with LBP) specifically neutralized the cofactor activity of the LBP preparation in a concentration-dependent manner. Bovine serum and plasma, as well as the C4BP preparation, optimally supported the binding of a rabbit anti-C4BP antiserum to immobilized cardiolipin. The binding of a rabbit anti-AT antiserum to solid-phase cardiolipin was sustained by the thrombin-AT preparation and bovine serum, but neither by bovine plasma nor by native AT, thus reproducing the behavior of patient no. 6 ACA. Taking advantage of the restricted recognition by the latter ACA of a cofactor from bovine origin appearing upon clotting, we studied the generation of such activity in human plasma supplemented with bovine AT or bovine prothrombin before clotting. In these conditions, patient no. 6 antibody binding to cardiolipin required the addition of bovine AT, whereas addition of bovine prothrombin alone was ineffective. We therefore concluded that those ACA targeted bovine AT once it has been modified/cleaved by thrombin. These findings underline the wide heterogeneity of ACA and the links that may exist between various coagulation pathways, inflammation and the complement system.
Physical Interaction Between Retinoic Acid Receptor and Sp1: Mechanism for Induction of Urokinase by Retinoic Acid
Authors: Suzuki Yasuhiro , Shimada Jun , Shudo Koichi , Matsumura Masatoshi , Crippa Massimo P. , Kojima Soichi
Published: October 2019

Induction of urokinase plasminogen activator (uPA) by retinoic acid (RA) is the initial event preceding certain subsequent biological changes in vascular endothelial cells. We investigated the molecular mechanism by which RA stimulates the expression of uPA, which lacks a canonical RA receptor (RAR)-responsive element, in bovine and human aortic endothelial cells. Upon stimulation with RA, mRNA levels of RAR and β transiently increased in parallel with the induction of uPA, and this increase was inhibited by cycloheximide. Results of transient transfection of RAR/RXR cDNAs and experiments using specific agonists and antagonists suggested that uPA induction is dependent upon RAR (initially, RAR) with the help of RXR. Deletion analysis of the uPA promoter suggested that RAR/RXR acts on GC box region within the uPA promoter. This was further supported by inhibition of Sp1 binding to this region. Coimmunoprecipitation studies, glutathioneS-transferase pull-down experiment, and mammalian two-hybrid assays suggested a physical interaction between RAR/RXR and Sp1. Furthermore, gel shift studies showed that the binding of Sp1 to the uPA GC box is significantly potentiated in the presence of RARs/RXRs. Finally, Sp1 and RAR/RXR synergistically enhanced the transactivation activity of the uPA promoter. These results suggest that (1) RA induces RARs mainly via RAR and that (2) RAR/RXR physically and functionally interact with Sp1, resulting in a potentiation of uPA transcription.
Interleukin-9 Regulates NF-κB Activity Through BCL3 Gene Induction
Authors: Richard Mélisande , Louahed Jamila , Demoulin Jean-Baptiste , Renauld Jean-Christophe
Published: October 2019

BCL3 encodes a protein with close homology to IκB proteins and interacts with p50 NF-κB homodimers. However, the regulation and transcriptional activity of BCL3 remain ill-defined. We observed here that interleukin-9 (IL-9) and IL-4, but not IL-2 or IL-3, transcriptionally upregulated BCL3 expression in T cells and mast cells. BCL3 induction by IL-9 was detected as soon as 4 hours after stimulation and appeared to be dependent on the Jak/STAT pathway. IL-9 stimulation was associated with an increase in p50 homodimers DNA binding activity, which was mimicked by stableBCL3 expression. This contrasts with tumor necrosis factor (TNF)-dependent NF-κB activation, which occurs earlier, involves p65/p50 dimers, and is dependent on IκB degradation. Moreover, IL-9 stimulation or BCL3 transient transfection similarly inhibited NF-κB–mediated transcription in response to TNF. Taken together, our observations show a new regulatory pathway for the NF-κB transcription factors through STAT-dependent upregulation ofBCL3 gene expression.
The Human (ΨL+μ−) proB Complex: Cell Surface Expression and Biochemical Structure of a Putative Transducing Receptor
Authors: Lemmers Bénédicte , Gauthier Laurent , Guelpa-Fonlupt Valérie , Fougereau Michel , Schiff Claudine
Published: October 2019

The surrogate light chain (ΨL) associates with μ and Ig-Igβ chains to form the preB-cell receptor that plays a critical role in early B-cell differentiation. Discrepancies exist in human concerning the existence of ΨL+μ− proB cells and the biochemical structure of such a proB-cell complex remains elusive. Among new antihuman VpreB monoclonal antibodies (MoAbs), 5 of the γκ isotype bound to recombinant and native VpreB protein with high affinity. They recognized 4 discrete epitopes, upon which 2 were in the extra-loop fragment. Such MoAbs detected the ΨL at the cell surface of either preB or on both proB and preB cells. The previously reported SLC1/SLC2 MoAbs recognize a conformational epitope specific for the μ/ΨL association in accordance with their preB-cell reactivity. Using the proB/preB 4G7 MoAb, ΨL cell surface expression was detected on normal bone marrow, not only on CD34−CD19+ preB but also on CD34+CD19+ proB cells. Futhermore, this MoAb identified ΨL+μ− fresh proB leukemic cells of the TEL/AML1 type. Biochemical studies showed that, at the proB stage, the ΨL is associated noncovalently with two proteins of 105 and 130 kD. Triggering of this complex induces intracellular Ca2+ flux, suggesting that the ΨL may be involved in a new receptor at this early step of the B-cell differentiation.
Lineage-Specific Modulation of Calcium Pump Expression During Myeloid Differentiation
Authors: Launay Sophie , Giannı̀ Maurizio , Kovàcs Tünde , Bredoux Raymonde , Bruel Arlette , Gélébart Pascal , Zassadowski Fabien , Chomienne Christine , Enouf Jocelyne , Papp Béla
Published: October 2019

Calcium is accumulated from the cytosol into the endoplasmic reticulum by sarco-endoplasmic reticulum calcium transport ATPase (SERCA) enzymes. Because calcium stored in the endoplasmic reticulum is essential for cell growth, differentiation, calcium signaling, and apoptosis and because different SERCA enzymes possess distinct functional characteristics, in the present report we explored SERCA expression during in vitro differentiation of the human myeloid/promyelocytic cell lines HL-60 and NB4 and of freshly isolated acute promyelocytic leukemia cells. Two SERCA species have been found to be coexpressed in these cells: SERCA 2b and another isoform, SERCAPLIM, which is recognized by the PLIM430 monoclonal antibody. Induction of differentiation along the neutrophil granulocytic lineage by all-trans retinoic acid or cyclic AMP analogs led to an increased expression of SERCAPLIM, whereas the expression of the SERCA 2b isoform was decreased. The modulation of SERCA expression was manifest also on the mRNA level. Experiments with retinoic acid receptor isoform-specific retinoids indicated that SERCA expression is modulated by retinoic acid receptor -dependent signaling. SERCA expression of retinoic acid-resistant cell variants was refractory to treatment. Differentiation along the monocyte/macrophage lineage by phorbol ester resulted in an increased expression of both SERCA isoforms. In addition, when cells were treated by phorbol ester in the presence of the glucocorticoid dexamethasone, a known inhibitor of monocyte differentiation, a selective blockage of the induction of SERCAPLIM was observed. Altered SERCA expression modified the functional characteristics of calcium transport into the endoplasmic reticulum. These observations show for the first time that the modulation of calcium pump expression is an integral component of the differentiation program of myeloid precursors and indicate that a lineage-specific remodelling of the endoplasmic reticulum occurs during cell maturation. In addition, these data show that SERCA isoforms may serve as useful markers for the study of myeloid differentiation.
Transforming Growth Factor-β1 Polarizes Murine Hematopoietic Progenitor Cells to Generate Langerhans Cell-Like Dendritic Cells Through a Monocyte/Macrophage Differentiation Pathway
Authors: Zhang Yi , Zhang Yan-yun , Ogata Masafumi , Chen Pan , Harada Akihisa , Hashimoto Shin-ichi , Matsushima Kouji
Published: October 2019

We have recently demonstrated that CD11b−/dullCD11c+ and CD11b+hiCD11c+ dendritic cell (DC) precursor subsets represent two distinct DC differentiation pathways from murine bone marrow lineage-phenotype negative (Lin−)c-kit+ hematopoietic progenitor cells (HPCs) stimulated with granulocyte-macrophage colony-stimulating factor (GM-CSF) + stem cell factor (SCF) + tumor necrosis factor  (TNF). We show here that transforming growth factor-β1 (TGF-β1) significantly inhibits the generation of these CD11b−/dullCD11c+ and CD11b+hiCD11c+ DC precursors. Phenotypically, this inhibitory effect was accompanied by markedly suppressed expression of Ia and CD86 antigens as well as major histocompatibility complex (MHC) class II transactivator (CIITA) and CC-chemokine receptor 7 (CCR7) mRNAs in Lin−c-kit+ HPC cultures stimulated with GM-CSF + SCF + TNF at day 6. TGF-β1 could also suppress mature DC differentiation from CD11b+hiCD11c+ DC precursors, but not the differentiation from CD11b−/dullCD11c+ DC precursors. In the absence of TNF, TGF-β1 markedly suppressed the expression of CIITA and CCR7 mRNAs in GM-CSF + SCF-stimulated Lin−c-kit+ HPCs at either day 6 or day 12 and induced the differentiation solely into monocytes/macrophages as evident in morphology, active phagocytic, and endocytic activities. These cells expressed high levels of F4/80 and E-cadherin antigens, but low or undetectable levels of Ia, CD86, and CD40 molecules. However, upon the stimulation with TNF + GM-CSF, these cells could further differentiate into mature DCs expressing high levels of Ia and E-cadherin, characteristics for Langerhans cells (LCs), and gained the capacity of enhancing allogenic MLR. Taken together, all of these findings suggest that TGF-β1 polarizes murine HPCs to generate LC-like DCs through a monocyte/macrophage differentiation pathway.
A Histomorphometric Evaluation of Heparin-Induced Bone Loss After Discontinuation of Heparin Treatment in Rats
Authors: Shaughnessy Stephen G. , Hirsh Jack , Bhandari Mohit , Muir Jeffrey M. , Young Edward , Weitz Jeffrey I.
Published: October 2019

Although it is well established that long-term heparin therapy causes osteoporosis, it is unknown whether heparin-induced bone loss is reversible when heparin treatment is stopped. To address this question, we randomized rats to once daily subcutaneous injections of either unfractionated heparin (1.0 U/g or 0.5 U/g) or saline for 28 days and then followed the rats for an additional 28 days off treatment. Based on histomorphometric analysis of the distal third of the right femur proximal to the epiphyseal growth plate, 1.0 U/g heparin caused a 30% loss in cancellous bone volume over the first 28 days. This was accompanied by a 137% increase in osteoclast surface and a 60% decrease in both osteoblast and osteoid surface. One month after cessation of heparin treatment, no recovery in these parameters was observed. Similarly, serum levels of alkaline phosphatase, a biochemical marker of bone formation, which continued to decrease over the course of heparin treatment, showed no signs of recovery in the subsequent 28 days off treatment. To explore the mechanism responsible for the prolonged effect of heparin on bone, we repeated the experiment giving 125I-labeled heparin in place of unlabeled heparin.125I-labeled heparin was found to accumulate in bone during the course of its administration, and be retained in bone for at least 56 days after stopping heparin treatment. These findings suggest that heparin-induced osteoporosis is not rapidly reversible because heparin is sequestered in bone for an extended period.
Molecular Analysis of 11q13 Breakpoints in Multiple Myeloma
Authors: Ronchetti Domenica , Finelli Palma , Richelda Raffaella , Baldini Luca , Rocchi Mariano , Viggiano Luigi , Cuneo Antonio , Bogni Silvia , Fabris Sonia , Lombardi Luigia , Maiolo Anna Teresa , Neri Antonino
Published: October 2019

The t(11;14)(q13;q32) chromosomal translocation, which is the hallmark of mantle cell lymphoma (MCL), is found in approximately 30% of multiple myeloma (MM) tumors with a 14q32 translocation. Although the overexpression of cyclin D1 has been found to be correlated with MM cell lines carrying the t(11;14), rearrangements of theBCL-1/cyclin D1 regions frequently involved in MCL rarely occur in MM cell lines or primary tumors. To test whether specific 11q13 breakpoint clusters may occur in MM, we investigated a representative panel of primary tumors by means of Southern blot analysis using probes derived from MM-associated 11q13 breakpoints. To this end, we first cloned the breakpoints and respective germ-line regions from a primary tumor and the U266 cell line, as well as the germ-line region from the KMS-12 cell line. DNA from 50 primary tumors was tested using a large panel of probes, but a rearrangement was detected in only one case using the KMS-12 breakpoint probe. Our results confirm previous findings that the 11q13 breakpoints in MM are scattered throughout the 11q13 region encompassing the cyclinD1 gene, thus suggesting the absence of 11q13 breakpoint clusters in MM.
Identification of Cord Blood Dendritic Cells as an Immature CD11c− Population
Authors: Sorg Rüdiger V. , Kögler Gesine , Wernet Peter
Published: October 2019

Dendritic cells (DC) are the main stimulators of primary T-cell responses and, thus, probably play a role in the immune reactions after stem cell transplantation. Very little is known about DC in cord blood (CB) and about their potential involvement in the low incidence and severity of acute graft-versus-host disease after CB transplantation. Here, CBDC were identified as a HLA-DR+ cell population, lacking the CD3, CD11b, CD14, CD16, CD19, CD34, CD56, and glycophorin A lineage markers (lin). This lin−/HLA-DR+population represented 0.3% ± 0.1% (mean ± SD; range, 0.1% to 0.6%; n = 15) of CB mononuclear cells, and CB contained 5.4 ± 3.2 × 103 CBDC/mL (1.8 to 13.0 × 103; n = 15). CBDC expressed CD4, CD11a, CD18, CD45RA, CD50, CD54, and CD123, but showed no expression of CD1a, CD11c, CD33, CD40, CD45R0, CD80, CD83, and CD86 and only limited expression of CD58, CD102, and CD116. Despite this immature phenotype, immunomagnetically lin−-enriched CBDC were potent stimulators of allogeneic CB T cells. As few as 266 ± 107 (193 to 530; n = 10) lin−/HLA-DR+ CBDC stimulated a significant response. However, CBDC failed to take up protein or peptide antigens. Thus, in CB there is a prevalence of a DC subpopulation, resembling the CD11c− DC identified in tonsils, the so-called plasmacytoid T cells, which may exert a function distinct from the CD11c+ DC subpopulation.