bluuebunny/crossref_metadata_2025 huggingface dataset:
Localization and Regulation of Thrombopoietin mRNA Expression in Human Kidney, Liver, Bone Marrow, and Spleen Using In Situ Hybridization
Authors: Sungaran R. , Markovic B. , Chong B.H.
Published: October 2019

Thrombopoietin (TPO) is the primary hematopoietic growth factor involved in the regulation of platelet production. Although the kidney, liver, bone marrow (BM), and spleen have been identified as the major sources of TPO production, the precise cellular location of TPO mRNA expression in these tissues remains unknown. We have identified the cells expressing TPO mRNA in the human kidney, liver, and BM using an in situ hybridization assay. In the BM of individuals with normal platelet counts, the hybridization signal was too weak to allow identification of the TPO mRNA expressing cells. However, in thrombocytopenic subjects with aplastic anemia, postchemotherapy marrow aplasia, and immune thrombocytopenia, the stromal cells showed strong TPO mRNA expression. In the human subjects with normal platelet counts, the cells of the proximal convoluted tubules of the kidney showed consistent positive staining whereas the signal in the cells of the distal convoluted tubules was less consistent. Strong hybridization signal was also evident in the hepatocytes. The hybridization signal in the spleen, even in thrombocytopenic subjects, was too weak to allow confident identification of the cells expressing TPO mRNA. In all subjects, the interstitial cells and endothelial cells of the liver and spleen, the renal peritubular cells, and the hematopoietic precursor cells of the BM showed no TPO mRNA expression. Our data suggest that TPO mRNA expression in the human BM may be modulated by platelet mass.
Clonality Analysis of Hematopoiesis in Essential Thrombocythemia: Advantages of Studying T Lymphocytes and Platelets
Authors: El-Kassar Nahed , Hetet Gilles , Brière Jean , Grandchamp Bernard
Published: October 2019

Essential thrombocythemia (ET) is a myeloproliferative disorder characterized by a sustained elevation of the platelet count in the absence of other causes of thrombocytosis. ET is difficult to diagnose, and the demonstration of clonal hematopoiesis may be of value. However, clonality analysis of hematopoietic cells based on the study of the X-chromosome inactivation pattern is complicated by the observation that some normal females present skewed lyonization. Moreover, DNA methylation of X-linked genes in hematopoietic cells may differ from that in other tissues. Appropriate controls for skewed lyonization are therefore critical for the study of clonality. We developed two techniques based on X-chromosome inactivation and polymerase chain reaction (PCR) analysis of polymorphisms, to study clonality in ET patients. Reverse transcriptase-PCR analysis of IDS, P55, and G6PD mRNAs was used to examine the different hematopoietic cell lineages including platelets in patients heterozygous for these polymorphisms and analysis of the HUMARA gene methylation pattern permitted us to study clonality in all nucleated cell fractions of the other patients. Using both types of assay and T lymphocytes as a control tissue for lyonization, clonal hematopoiesis was demonstrated in 28 patients. In 14 patients, the granulocytes were polyclonal; among these patients, platelets were monoclonal in 3 cases, polyclonal in 7 cases, and in the remaining 4 cases this fraction could not be studied because the patients were homozygotes for all RNA markers. No conclusion about clonality could be drawn in 6 cases. Polyclonal hematopoiesis was found in all the cases of reactive thrombocytosis. These findings confirm the high frequency of monoclonal hematopoiesis in ET, the utility of studying platelets, and the possibility of using T lymphocytes as a control tissue for X-chromosome inactivation patterns.
Antisense Inhibition of c-fes Proto-oncogene Blocks PMA-Induced Macrophage Differentiation in HL60 and in FDC-P1/MAC-11 Cells
Authors: Manfredini Rossella , Balestri Raffaella , Tagliafico Enrico , Trevisan Francesca , Pizzanelli Michela , Grande Alexis , Barbieri Daniela , Zucchini Patrizia , Citro Gennaro , Franceschi Claudio , Ferrari Sergio
Published: October 2019

To gain some insight into the role of c-fes in macrophage differentiation, we have analyzed the ability of HL60 leukemic promyelocytic cells and FDC-P1/MAC-11 murine myeloid precursor cells to differentiate in response to phorbol esters after inhibition of c-fes function. Fes inactivation has been obtained by using oligodeoxynucleotides (ODN) complementary to the 5′ region of c-fes mRNA and to 5′ splice junctions of c-fes primary transcript. After 5 days (d) in culture, in several separate experiments performed with different ODN preparations, a complete inhibition of c-fes expression was observed in HL60 and in FDC-P1/MAC-11 cells. No perturbation of cell growth was evident in our experimental conditions in both cell lines after c-fes inhibition. Furthermore, in HL60 cells lacking c-fes product, an almost complete downregulation of the α4β1 fibronectin receptor occurred. However, in both cell lines, the induction of macrophage differentiation by phorbol esters resulted in an almost complete maturation arrest as evaluated by morphological, cytochemical, immunological criteria, and by the cytofluorimetric cell cycle analysis. A loss of the adhesion capacity of both myeloid cell lines, when compared to terminally differentated macrophages, was also observed. These results suggest that HL60 and FDC-P1/MAC-11 cells, when treated with phorbol 12-myristate 13-acetate, require c-fes protein expression to activate the genetic program underlying macrophage differentiation.
Measurement of Spontaneous and Therapeutic Agent-Induced Apoptosis With BCL-2 Protein Expression in Acute Myeloid Leukemia
Authors: Banker Deborah E. , Groudine Mark , Norwood Tom , Appelbaum Frederick R.
Published: October 2019

We have designed in vitro assays to investigate the possible association between apoptosis and chemotherapeutic sensitivity in acute myeloid leukemias (AMLs). Consistent low levels of spontaneous apoptosis were observed in myeloid cells from normal bone marrow samples, while untreated cells collected from 56 de novo AML patients showed variable apoptosis. Control myeloid cells showed increased apoptosis after in vitro treatments with daunomycin (DNR), cytosine arabinoside (ARA-C), or gamma irradiation (RAD). Most AML samples showed less treatment-associated apoptosis, suggesting that apoptosis responses to therapeutic agents may be frequently attenuated in AML. Certain cytogenetic abnormalities common in AML may affect apoptosis, as acute promyelocytic leukemia (APL) samples with t(15; 17) karyotypes showed consistently low levels of spontaneous and treatment-associated apoptosis. Apoptosis assays may provide unique functional subtyping of AMLs, as other common cytogenetic subsets showed variable apoptosis. Altered function of two well-characterized regulators of apoptosis, BCL-2 and p53, was not entirely responsible for this variability. A genomic p53 mutation was found in only one AML sample. All samples that demonstrated the highest BCL-2–positive cell fractions showed low apoptosis, but reduced apoptosis was seen in both the presence and absence of BCL-2 overexpression. Finally, data from matched diagnosis and relapse sample pairs suggest that neither further reduced apoptosis nor additional BCL-2 overexpression is necessarily associated with disease progression.
Interferon-β Interrupts Interleukin-6–Dependent Signaling Events in Myeloma Cells
Authors: Berger Lloyd C. , Hawley Robert G.
Published: October 2019

Type I interferons (IFNs-α and IFN-β) bind to a common receptor to exert strong antiproliferative activity on a broad range of cell types, including interleukin-6 (IL-6)–dependent myeloma cells. In this study, we investigated the effect of IFN-β pretreatment on IL-6–stimulated mitogenic signaling in the human myeloma cell line U266. IL-6 induced transient tyrosine phosphorylation of the IL-6 receptor signal-transducing subunit gp130, the gp130-associated protein tyrosine kinases Jak1, Jak2, and Tyk2, the phosphotyrosine phosphatase PTP1D/Syp, the adaptor protein Shc and the mitogen-activated protein kinase Erk2, and accumulation of GTP-bound p21ras. Prior treatment of U266 cells with IFN-β downregulated IL-6–induced tyrosine phosphorylation of gp130, Jak2, PTP1D/Syp, Shc, and Erk2, and GTP-loading of p21ras. Further analysis indicated that treatment with IFN-β disrupted IL-6–induced binding of PTP1D/Syp to gp130 and the adaptor protein Grb2; IFN-β pretreatment also interfered with IL-6–induced interaction of Shc with Grb2 and a 145-kD tyrosine-phosphorylated protein. These results suggest a novel mechanism whereby type I IFNs interrupt IL-6–promoted mitogenesis of myeloma cells in part by preventing the formation of essential signaling complexes leading to p21ras activation.
Deletions and Loss of Expression of P16INK4a and P21Waf1 Genes Are Associated With Aggressive Variants of Mantle Cell Lymphomas
Authors: Pinyol Magda , Hernandez Luis , Cazorla Maite , Balbı́n Milagros , Jares Pedro , Fernandez Pedro L. , Montserrat Emilio , Cardesa Antonio , Lopez-Otı́n Carlos , Campo Elı́as
Published: October 2019

Mantle cell lymphoma (MCL) is molecularly characterized by bcl-1 rearrangement and cyclin D1 gene overexpression. Some aggressive variants of MCL have been described with blastic or large cell morphology, higher proliferative activity, and shorter survival. The cyclin-dependent kinase inhibitors (CDKIs) p21Waf1 and p16INK4a have been suggested as candidates for tumor-suppressor genes. To determine the role of p21Waf1 and p16INK4a gene alterations in MCLs, we examined the expression, deletions, and mutations of these genes in a series of 24 MCLs, 18 typical, and 6 aggressive variants. Loss of expression and/or deletions of p21Waf1 and p16INK4a genes were detected in 4 (67%) aggressive MCLs but in none of the typical variants. Two aggressive MCLs showed a loss of p16INK4a expression. These cases showed homozygous deletions of p16INK4a gene by Southern blot analysis. An additional aggressive MCL in which expression could not be examined showed a hemizygous 9p12 deletion. Loss of p21Waf1 expression at both protein and mRNA levels was detected in an additional aggressive MCL. No p21Waf1 gene deletions or mutations were found in this case. The p21Waf1 expression in MCLs was independent of p53 mutations. The two cases with p53 mutations showed p21Waf1 and p16INK4a expression whereas the 4 aggressive MCLs with p16INK4a and p21Waf1 gene alterations had a wild-type p53. p21Waf1 and p16INK4a were expressed at mRNA and protein levels in all typical MCLs examined. No gene deletions or point mutations were found in typical variants. Two typical MCLs showed an anomalous single-stranded conformation polymorphism corresponding to the known polymorphisms at codon 148 of p16INK4a gene and codon 31 of p21Waf1 gene. These findings indicate that p21Waf1 and p16INK4a alterations are rare in typical MCLs but the loss of p21Waf1 and p16INK4a expression, and deletions of p16INK4a gene are associated with aggressive variants of MCLs, and they occur in a subset of tumors with a wild-type p53 gene.
Enhancement of Macrophage Colony-Stimulating Factor–Induced Growth and Differentiation of Human Monocytes by Interleukin-10
Authors: Hashimoto Shin-ichi , Yamada Muneo , Motoyoshi Kazuo , Akagawa Kiyoko S.
Published: October 2019

Interleukin-10 (IL-10) has been reported to be a negative cytokine for monocytes/macrophages. In the present study, we showed that IL-10 is rather a positive cytokine and augments the growth and differentiation of human monocytes stimulated with macrophage colony-stimulating factor (M-CSF ). Highly purified adherent human monocytes were cultured for 7 days with M-CSF in the presence or absence of IL-10. The number of recovered cells increased in the culture of monocytes with M-CSF + IL-10 compared to the culture with M-CSF alone. IL-10 alone was not enough to maintain the survival and differentiation of monocytes into macrophages. Morphological change cultured in M-CSF was also accelerated by addition of IL-10, and macrophages cultured in M-CSF + IL-10 were more elongated compared to macrophages cultured with M-CSF alone. Binding of 125I-M-CSF to monocytes incubated with M-CSF + IL-10 was about 1.7-fold higher than that to monocytes incubated with M-CSF alone. In accordance with the binding study, Northern blot analysis showed that the levels of the expression of c-fms, M-CSF receptor, mRNA in macrophages cultured in M-CSF + IL-10 were higher than that in macrophages cultured in M-CSF alone. Macrophages cultured in M-CSF + IL-10 expressed higher level of FcγRI, II, III, and showed augmented Fcγ receptor mediated phagocytosis. The former also produced higher level of H2O2 and O−2 , when stimulated with zymosan, and of IL-6 when stimulated with lipopolysaccharide compared to the latter. These results taken together suggest that IL-10 augments the growth and differentiation of human monocytes cultured in M-CSF.
Erythropoietin Activates Raf1 by an Shc-Independent Pathway in CTLL-EPO-R Cells
Authors: Barber Dwayne L. , Corless Cristin N. , Xia Kai , Roberts Thomas M. , D'Andrea Alan D.
Published: October 2019

Stimulation of the erythropoietin receptor (EPO-R) or the interleukin-2 receptor (IL-2-R) by their respective ligands has been reported to activate tyrosine phosphorylation of the cytoplasmic protein, Shc. We have recently characterized a cell line, CTLL-EPO-R, that contains functional cell-surface receptors for both EPO and IL-2. Although stimulation with IL-2 or IL-15 resulted in the rapid, dose-dependent tyrosine phosphorylation of Shc, stimulation with EPO failed to activate Shc. EPO, IL-2, and IL-15 activated the tyrosine phosphorylation of the adaptor protein, Shp2, and the association of Shp2/Grb2/cytokine receptor complexes. In addition, EPO, IL-2, and IL-15 activated Raf1 and ERK2, demonstrating that the Raf1/MEK/MAP kinase pathway was activated. These results indicate that multiple biochemical pathways are capable of conferring a mitogenic signal in CTLL-EPO-R. EPO can activate the Raf1/MEK/ MAP kinase pathway via Shc-dependent or Shc-independent pathways, and Shc activation is not required for EPO-dependent cell growth in CTLL-EPO-R.
Fas-Mediated Apoptosis of the Hematopoietic Progenitor Cells in Mice Infected With Murine Cytomegalovirus
Authors: Mori Takehiko , Ando Kiyoshi , Tanaka Kazuo , Ikeda Yasuo , Koga Yasuhiro
Published: October 2019

The effects of cytomegalovirus (CMV) infection on hematopoietic progenitor cells in vivo were investigated to elucidate the pathogenesis of CMV-induced myelosuppression. BALB/c mice were inoculated with 0.2LD50 of murine CMV (MCMV). Lineage marker negative, c-kit positive (Lin−c-kit+) and Lin−CD34+ cells, which are both phenotypically defined as hematopoietic progenitor cells, showed a significant reduction in number on day 3 postinfection (pi). Moreover, the reduction in the number of day-14 colony-forming units-spleen (CFU-S), another indicator to identify hematopoietic progenitor cells, was noted on day 3 pi. To clarify the mechanism of such depletion, we examined the cells undergoing apoptosis in the Lin− populations and found a 15-fold increase in the apoptosis-induction of these cells. Furthermore, an increase in the expression level of Fas, which mediates apoptosis, was observed in such Lin−c-kit+ and Lin−Sca-1+ cells on day 3 pi. In vitro treatment with the anti-Fas antibody accelerated the apoptosis in Lin− cells, but not in the uninfected control cells, thus indicating that the upregulated Fas on Lin− cells is directly related to the acceleration of apoptosis found in these cells in vivo. These results suggest that MCMV infection reduces the number of hematopoietic progenitor cells in bone marrow at least in part due to Fas-mediated apoptosis, and this phenomenon is thus considered to contribute to CMV-induced myelosuppression.
Macrophage Inflammatory Protein-1α and Interferon-Inducible Protein 10 Inhibit Synergistically Induced Growth Factor Stimulation of MAP Kinase Activity and Suppress Phosphorylation of Eukaryotic Initiation Factor 4E and 4E Binding Protein 1
Authors: Aronica Susan M. , Gingras Anne-Claude , Sonenberg Nahum , Cooper Scott , Hague Nancy , Broxmeyer Hal E.
Published: October 2019

Granulocyte-macrophage colony-stimulating factor (GM-CSF ) and Steel factor (SLF ) synergistically stimulate Raf-1 kinase activity, protein synthesis, and proliferation in hematopoietic MO7e cells; synergistic action of these factors is blocked by the suppressive chemokines macrophage inflammatory protein-1α (MIP-1α) and interferon-inducible protein 10 (IP-10; Aronica et al, J Biol Chem 270:21998, 1995). We assessed the potential for both stimulatory and inhibitory factors to act through the MAP kinase signaling pathway by studying the effects of growth factors and chemokines on MAP kinase activation. Also, because activation of kinase signaling pathways and stimulation of protein synthesis by peptide growth factors are associated with increased phosphorylation of eukaryotic initiation factor 4E (eIF-4E) and the translational repressor 4E-binding protein 1 (4E-BP1) in some target cells, we investigated whether growth factor treatment could alter eIF-4E or 4E-BP1 phosphorylation state in MO7e cells. We report that treatment of MO7e cells with GM-CSF and SLF stimulated significant, greater-than-additive increases in MAP kinase activity and the phosphorylation of both eIF-4E and 4E-BP1. Increased 4E-BP1 phosphorylation correlated with a decrease in the association of 4E-BP1 with eIF-4E. Growth factor-induced phosphorylation of 4E-BP1 and dissociation of 4E-BP1 from eIF-4E was blocked in cells treated with rapamycin, wortmannin, or PD098059. Treatment of cells with IP-10 or MIP-1α blocked the stimulatory effects of GM-CSF and SLF, resulting in suppression of MAP kinase activity, eIF-4E and 4E-BP1 phosphorylation, and eIF-4E/4E-BP1 dissociation. Our results suggest that GM-CSF and SLF exert part of their combined growth-promoting effects on MO7e cells through activation of MAP kinase and enhancement of eIF-4E and 4E-BP1 phosphorylation and dissociation and that suppression of growth factor-induced protein synthesis by MIP-1α and IP-10 involves translational repression at the level of eIF-4E.
Phenotypic and Functional Changes Induced at the Clonal Level in Hematopoietic Stem Cells After 5-Fluorouracil Treatment
Authors: Randall Troy D. , Weissman Irving L.
Published: October 2019

A significant fraction of hematopoietic stem cells (HSCs) have been shown to be resistant to the effects of cytotoxic agents such as 5-fluorouracil (5-FU), which is thought to eliminate many of the rapidly dividing, more committed progenitors in the bone marrow and to provide a relatively enriched population of the most primitive hematopoietic progenitor cells. Although differences between 5-FU–enriched progenitor populations and those from normal bone marrow have been described, it remained unclear if these differences reflected characteristics of the most primitive stem cells that were revealed by 5-FU, or if there were changes in the stem-cell population itself. Here, we have examined some of the properties of the stem cells in the bone marrow before and after 5-FU treatment and have defined several activation-related changes in the stem-cell population. We found that long-term reconstituting stem cells decrease their expression of the growth factor receptor c-kit by 10-fold and increase their expression of the integrin Mac-1 (CD11b). These changes begin as early as 24 hours after 5-FU treatment and are most pronounced within 2 to 3 days. This activated phenotype of HSCs isolated from 5-FU–treated mice is similar to the phenotype of stem cells found in the fetal liver and to the phenotype of transiently repopulating progenitors in normal bone marrow. We found that cell cycle is induced concomitantly with these physical changes, and within 2 days as many as 29% of the stem-cell population is in the S/G2/M phases of the cell cycle. Furthermore, when examined at a clonal level, we found that 5-FU did not appear to eliminate many of the transient, multipotent progenitors from the bone marrow that were found to be copurified with long-term repopulating, activated stem cells. These results demonstrate the sensitivity of the hematopoietic system to changes in its homeostasis and correlate the expression of several important surface molecules with the activation state of HSCs.
Immature Human Megakaryocytes Produce Nuclear-Associated Acetylcholinesterase
Authors: Lev-Lehman Efrat , Deutsch Varda , Eldor Amiram , Soreq Hermona
Published: October 2019

Acetylcholinesterase (AChE) is expressed in murine megakaryocytes (MK), where its antisense inhibition suppresses differentiation, yet was never detected in human MK. Here, we report that AChE is produced in normal human bone marrow MK and in cell lines derived thereof. Reverse transcriptase-polymerase chain reaction (RT-PCR) amplification showed two ACHEmRNA forms in human megakaryoblastic DAMI cells. In situ hybridization demonstrated ACHEmRNA surrounding the nucleus of small DAMI cells and the nuclear lobes of large, polyploid cells. Differentiation induction with phorbol ester and exposure to recombinant human thrombopoietin suppressed both ACHEmRNA and AChE activity. The residual AChE in mature differentiated cells acquired higher stability and detergent-sensitivity as compared with AChE in small proliferating cells. AChE activity was primarily associated with nuclei of both DAMI cells and small (10 μm) primary proliferating human bone marrow MK identified with GPIIb/IIIa antibodies. This activity was significantly reduced in medium size MK (10 to 25 μm) and was almost undetectable in large MK (<25 μm), yet was twofold more abundant in some large MK from idiopathic thrombocytopenia purpura (ITP) patients with accelerated MK maturation. The loss of AChE activity at the transition from proliferating to differentiating MK highlights species-specific differences in its expression, suggesting a distinct role for AChE in human MK development.
The Inhibitor Antibody Response Is More Complex in Hemophilia A Patients Than in Most Nonhemophiliacs With Factor VIII Autoantibodies
Authors: Prescott Richard , Nakai Hiroaki , Saenko Evgueni L. , Scharrer Inge , Nilsson Inga Marie , Humphries John E. , Hurst Deborah , Bray Gordon , Scandella Dorothea ,   the Recombinate and Kogenate Study Groups
Published: October 2019

Approximately 25% of hemophilia A patients infused with factor VIII (fVIII) mount an immune response, which leads to its inactivation. Anti-fVIII autoantibodies are also seen rarely in individuals with normal fVIII. We have previously demonstrated that some anti-A2 and anti-C2 domain antibodies are fVIII inhibitors and that many patients have additional inhibitors with a fVIII light chain (LCh) epitope outside C2. Because the contribution of the different antibodies to the plasma inhibitor titer had been examined in a limited number of patients (14), we report in this study a more extensive analysis of 55 plasmas. The dominant inhibitors in 62% (13 of 21) of autoantibody plasmas were directed only against C2 or A2, but not both, whereas this pattern was found in only 15% (5 of 34) of hemophilic plasmas. In addition, anti-A2 inhibitors were present in 71% (24 of 34) of hemophilic plasmas, but only 33% (7 of 21) of autoantibody plasmas. These results demonstrated that the inhibitor response in hemophiliacs was more complex and the epitope specificity was somewhat different. A comparison of hemophiliacs treated only with plasma fVIII or recombinant fVIII showed no significant differences in the complexity of the inhibitor response, as ≥ 2 different inhibitor antibodies were present in 78% (18 of 23) of the former and 82% (9 of 11) of the latter. In contrast, the major inhibitors in 35% (8 of 23) of hemophiliacs treated with plasma fVIII were directed against C2 and another LCh epitope within residues 1649-2137, but not A2, while none (0 of 11) treated with recombinant fVIII had this pattern.
Functional Characterization and Modulation of Cytokine Production by CD8+ T Cells from Human Immunodeficiency Virus-Infected Individuals
Authors: Maggi Enrico , Manetti Roberto , Annunziato Francesco , Cosmi Lorenzo , Giudizi Maria Grazia , Biagiotti Roberta , Galli Grazia , Zuccati Giuliano , Romagnani Sergio
Published: October 2019

CD8+ T-cell clones were generated from peripheral blood mononuclear cells (PBMC) of three human immunodeficiency virus (HIV)-seronegative individuals and six HIV-seropositive individuals and assessed for their cytokine secretion profile, cytolytic potential, and chemokine production. While the great majority of CD8+ T-cell clones generated from HIV-seronegative individuals produced interferon (IFN)-γ, but not interleukin-4 (IL-4), that is a type 1 cytotoxic (Tc1) profile, high numbers of CD8+ T-cell clones generated from HIV-seropositive individuals produced IL-4 in addition to IFN-γ or IL-4 alone, thus showing a type 0 cytotoxic (Tc0)- or a type 2 cytotoxic (Tc2) profile, respectively. Tc0/Tc2 cells displayed lower cytolytic activity than Tc1 cells, including a reduced ability to lyse autologous targets pulsed with HIV or HIV peptides. By contrast, the production of chemokines RANTES and macrophage inflammatory protein-1α was comparable in Tc1, Tc0, and Tc2 clones irrespective of whether they were derived from HIV-seronegative or HIV-seropositive individuals. When CD8+ T-cell clones were generated from PBMC cultures of HIV-seronegative individuals conditioned with IL-4 plus an anti–IL-12 antibody (Ab), a shift towards the Tc0/Tc2-like profile was observed. Conversely, the addition to PBMC cultures of IL-12 plus an anti – IL-4 Ab shifted the differentiation of CD8+ T cells from HIV-infected individuals towards the Tc1-like profile, whereas IL-12 or anti–IL-4 Ab alone had a lower Tc1-promoting effect. Irradiated PBMC from HIV-infected individuals, used as feeder cells, shifted the differentiation of CD8+ T cells from a healthy HIV-seronegative individual towards the Tc0/Tc2-like profile. On the other hand, a shift towards the Tc1-like profile was noted in CD8+ T-cell clones generated from the skin specimens of two HIV-seropositive patients with Kaposi's sarcoma, successfully treated with IFN-α, in comparison to CD8+ clones generated from the same skin areas before treatment. The IFN-α–induced Tc1 shift could be prevented by the incubation of skin-infiltrating CD8+ T cells with IL-4 before cloning. Taken together, these data indicate that both defective production of IL-12 and abnormal IL-4 production in bulk PBMC populations of HIV-infected individuals may contribute to the development of high numbers of CD8+ T-cell clones showing a Tc0/Tc2-like phenotype and reduced cytolytic potential against HIV itself. They also suggest that the cytokine profile of CD8+ T-cell clones can be modulated by cytokines (or anticytokine Ab) both in vitro and in vivo.
Reed-Sternberg Cells of Classical Hodgkin's Disease React With the Plasma Cell-Specific Monoclonal Antibody B-B4 and Express Human Syndecan-1
Authors: Carbone Antonino , Gloghini Annunziata , Gattei Valter , Degan Massimo , Improta Salvatore , Aldinucci Donatella , Canzonieri Vincenzo , Perin Tiziana , Volpe Rachele , Gaidano Gianluca , Zagonel Vittorina , Pinto Antonio
Published: October 2019

Although the cellular origin of Reed-Sternberg (RS) cells of classical Hodgkin's disease (HD) has been a controversial issue for many years, recent immunophenotypic and molecular studies have suggested that RS cells of a subset of classical HD cases may be related to B cells. To further define the immunophenotypic features and the differentiation stage of RS cells, a series of 56 HD samples, including both nodular lymphocyte predominance (LP) (eight cases) and classical HD (nodular sclerosis [NS], 32 cases; mixed cellularity [MC], 16 cases) with a non-T–cell phenotype, were evaluated for the immunohistochemical expression of the B-B4 antigen, a specific marker for terminally differentiated B cells. Because the cDNA of the B-B4 antigen encodes syndecan-1, a member of a family of transmembrane heparan sulfate proteoglycans thought to be involved in binding cells of the B lineage to the interstitial matrix, the B-B4 immunoreactivity was correlated with the expression of syndecan-1 in HD-derived cell lines (L428, KM-H2), as detected by both reverse transcriptase polymerase chain reaction (RT-PCR) studies and Western blotting. Our results show that B-B4 reacts with RS cells and their morphological variants of all cases of classical HD, irrespective of their antigenic phenotype (B, undetermined), albeit at a varying degree of cellular expression. Notably, a high reactivity and staining intensity for the B-B4 monoclonal antibody (MoAb) was restricted to tumor cells from NS HD. In cases of the latter subtype, B-B4 positivity was also found in sclerosis-trapped spindle cells (fibrocytes/fibroblasts). Conversely, the putative tumor cells of nodular LP HD were consistently unreactive with the B-B4 MoAb. Finally, we have demonstrated by RT-PCR, flow cytometry, and Western blotting that cultured RS cells, of B and undetermined phenotype, express syndecan-1 mRNA and produce a form of syndecan-1, recognized by the B-B4 MoAb, which is predominantly associated with glycosaminoglycans and is present at the cell surface. Our detection of the plasma cell-specific antigen B-B4 (syndecan-1) on tumor cells of classical HD further supports that RS cell progenitors may be related to germinal/postgerminal center mature B cells and suggests that expression of syndecan-1 may contribute to some of the typical biologic and histopathologic features of classical HD, with a special regard to the NS subtype.
Expression of the Multidrug Resistance-Associated Protein Gene in Refractory Lymphoma: Quantitation by a Validated Polymerase Chain Reaction Assay
Authors: Zhan Zhirong , Sandor Victor A. , Gamelin Erick , Regis Joanna , Dickstein Bruce , Wilson Wyndham , Fojo Antonio T. , Bates Susan E.
Published: October 2019

Previous work investigating the role of MDR-1 overexpression in relapsed and refractory lymphoma led us to investigate a possible role for multidrug resistance-associated protein (MRP) as a cause of resistance in patients who did not overexpress MDR-1. A quantitative polymerase chain reaction (PCR) method for measuring MRP expression was validated. Immunoblot analysis suggested that no major discrepancy was present between mRNA expression and protein levels. MRP levels were found to be independent of sample tumor content by immunophenotyping, suggesting that the presence of normal cells had no significant impact on measurements of MRP expression. We evaluated MRP in 55 biopsy samples from 40 patients with refractory lymphoma enrolled on a trial of infusional chemotherapy (EPOCH). Pre- and post-EPOCH samples were available from 15 patients. MRP levels were also evaluated in 16 newly diagnosed, untreated lymphoma patient samples. No significant difference in MRP mRNA expression was noted between pre- and post-EPOCH groups. Also, MRP levels in the newly diagnosed patient samples were not significantly different from either pre- or post-EPOCH groups. Two of 15 paired pre- and post-EPOCH patient samples exhibited overexpression of MRP after EPOCH chemotherapy, with measured increases of 10-fold and 18-fold. We conclude that MRP overexpression is not responsible for non–P-glycoprotein (Pgp)–mediated drug resistance in the majority of these patients, although it may be important in a subset of patients. Defining this subset prospectively could aid in the development of clinical trials of MRP modulation in drug-resistant lymphoma.
Neutrophils Can Adhere Via α4β1 -Integrin Under Flow Conditions
Authors: Reinhardt Paul H. , Elliott John F. , Kubes Paul
Published: October 2019

In this study we investigated the possibility that an alternative pathway exists for neutrophil recruitment, namely an α4β1 -dependent pathway. A parallel plate chamber was used to investigate whether neutrophils could tether, roll, and adhere to tumor necrosis factor α (TNFα)-stimulated endothelium via α4β1 . α4β1 -integrin was induced on neutrophils using dihydrocytochalasin B and either an endogenous (endothelial-derived) chemotactic agent or an exogenous chemotactic molecule. α4β1 -expressing neutrophils could stably adhere under shear force (2 dyne/cm2) to TNFα-stimulated endothelium independent of the β2 -integrin. The firm adhesion was entirely abolished by antibodies directed against either the α4 or β1 -integrin subunits. However, the rolling interaction was not dependent on α4β1 but was abolished by antiselectin therapy. Neutrophils expressing α4β1 could also tether to the endothelium in the presence of antiselectin therapy, but at shear stresses less than 2 dyne/cm2. α4β1 -expressing neutrophils also tethered to and stably adhered (no rolling) to VCAM-1– but not to ICAM-1–transfected L cells. The interaction only occurred at shear stress less than 2 dyne/cm2. A cell line (Ramos) known to express high quantities of α4β1 -integrin interacted with VCAM-1–transfected L cells at very similar shear conditions. α4β1 -expressing neutrophils were also able to adhere to a second α4 -integrin ligand, fibronectin; however, this interaction only occurred under static conditions. These data suggest that, under certain conditions, neutrophils can adhere independently of the β2 -integrin pathway and adhere via the α4β1 -integrin. This study refutes the concept that α4β1 -integrin adhesion is restricted to mononuclear leukocytes and is not functional on human neutrophils.
High Prevalence of Hepatitis G Virus in Bone Marrow Transplant Recipients and Patients Treated for Acute Leukemia
Authors: Skidmore Susan J. , Collingham Kathryn E. , Harrison Paul , Neilson Jeffrey R. , Pillay Deenan , Milligan Donald W.
Published: October 2019

Hepatitis G virus (HGV) is a newly described virus that has been implicated in transfusion-associated hepatitis. The prevalence of HGV in a group of multitransfused patients with hematological malignancy was studied using a reverse transcription polymerase chain reaction technique. Transfusion histories and serum aspartate aminotransferase (AST) levels were recorded. HGV was detected in 29 of 60 (48%) patients. There was no difference in HGV positivity rates between those with normal AST levels and those with raised AST levels. Analysis of patients by treatment type showed that 20 of 33 (61%) patients who received a bone marrow transplantation procedure were HGV positive compared with 9 of 27 (33%) treated with conventional combination chemotherapy (P = .036) despite similar transfusion histories. There was no significant difference in HGV positivity between patients treated before the introduction of United Kingdom blood donor screening for hepatitis C virus antibody:18 of 39 (46%) and those treated after the introduction of screening 11 of 21 (52%). HGV infection appears to be extremely common in these patients; however, the clinical significance of these findings with respect to liver dysfunction is not yet clear.
Prevention of Graft-Versus-Host Disease in Mice Using a Suicide Gene Expressed in T Lymphocytes
Authors: Cohen José L. , Boyer Olivier , Salomon Benoı̂t , Onclercq Rosine , Charlotte Frédéric , Bruel Sylvie , Boisserie Gilbert , Klatzmann David
Published: October 2019

Alloreactive T cells present in a bone marrow transplant are responsible for graft-versus-host disease (GVHD), but their depletion is associated with impaired engraftment, immunosuppression, and loss of the graft-versus-leukemia effect. We developed a therapeutic strategy against GVHD based on the selective destruction of these alloreactive T cells, while preserving a competent T-cell pool of donor origin. We generated transgenic mice expressing in their T lymphocytes the Herpes simplex type 1 thymidine kinase (TK) suicide gene that allows the destruction of dividing T cells by a ganciclovir treatment. T cells expressing the TK transgene were used to generate GVHD in irradiated bone marrow grafted mice. We show that a short 7-day ganciclovir treatment, initiated at the time of bone marrow transplantation, efficiently prevented GVHD in mice receiving TK-expressing T cells. These mice were healthy and had a normal survival. They maintained a T-cell pool of donor origin that responded normally to in vitro stimulation with mitogens or third party alloantigens, but were tolerant to recipient alloantigens. Our experimental system provides the proof of concept for a therapeutic strategy of GVHD prevention using genetically engineered T cells.
Leptin Stimulates Fetal and Adult Erythroid and Myeloid Development
Authors: Mikhail Adel A. , Beck Eric X , Shafer Alan , Barut Bruce , Smith Gbur Julie , Zupancic Thomas J. , Schweitzer Anthony C. , Cioffi Joseph A. , Lacaud Georges , Ouyang Bin , Keller Gordon , Snodgrass H. Ralph
Published: October 2019

The ob gene product, leptin, has been shown in several studies to be involved in weight control and recombinant leptin recently has entered clinical trials to treat obesity. The leptin receptor (OB-R/B219) is expressed in a variety of protein isoforms not only in the central nervous system, but also in reproductive, and hematopoietic tissues. We reported recently that the OB-R/B219 was associated with a variety of hematopoietic lineages as well as the small fraction of cells containing the long-term reconstituting hematopoietic stem cells. Herein we report that leptin significantly stimulates the proliferation and differentiation of yolk sac cells and fetal liver cells and stimulates directly hematopoietic precursors. Leptin alone can increase the number of macrophage and granulocyte colonies, and leptin plus erythropoietin act synergistically to increase erythroid development. These data show that leptin has a significant, direct effect on early hematopoietic development and can stimulate the differentiation of lineage-restricted precursors of the erythrocytic and myelopoietic lineages. These observations along with a recent report strongly support our previous hypothesis that leptin has an unanticipated important role in hematopoietic and immune system development.
Mouse Strain-Dependent Changes in Frequency and Proliferation of Hematopoietic Stem Cells During Aging: Correlation Between Lifespan and Cycling Activity
Authors: de Haan Gerald , Nijhof Willem , Van Zant Gary
Published: October 2019

We have quantified the frequency and proliferation of five subsets of primitive hematopoietic cells, using the cobblestone area forming cell (CAFC) assay, in marrow of five strains of mice with lifespans ranging from about 500 to 800 days. Stem cell characteristics were determined in young (6 weeks) and old (12 months) mice. We report striking effects of both intrinsic strain lifespan and individual mouse age on stem cell populations. First, the relative and absolute numbers of the most primitive stem cell subsets was threefold to fourfold higher in old than in young mice. Second, a considerable strain-to-strain variation in the number of primitive cells was observed: when absolute frequencies were calculated, there was a trend for longer lifespan to be correlated with a larger stem cell pool. Third, stem cells from old mice had a far lower cycling activity than cells from young mice. However, this was highly strain dependent: short-lived C3H/He and CBA/J mice showed a stronger reduction in cycling activity during aging than long-lived C57BL/6 mice. Finally, a significant negative correlation was demonstrated in young mice between maximal lifespan and proliferative activity. These data show that aging has a major impact on the frequency and cell-cycle kinetics of primitive hematopoietic cell compartments. In addition, the observation that cycling activity of stem cells is related to the maximal lifespan of the mouse strain may open ways to identify the genetic mechanisms of both strain- and age-dependent variation in the structure of primitive hematopoietic cell compartments.
Transgenic Mice Overexpressing Human c-mpl Ligand Exhibit Chronic Thrombocytosis and Display Enhanced Recovery From 5-Fluorouracil or Antiplatelet Serum Treatment
Authors: Zhou Wen , Toombs Christopher F. , Zou Tong , Guo Jane , Robinson Murray O.
Published: October 2019

The consequences of long-term in vivo expression of human c-mpl ligand in a mouse model were examined. Transgenic mice expressing the human full-length cDNA in the liver exhibited a fourfold increase in circulating platelet count that persisted stably over the life of the animals. Transgenic animals thrived and appeared healthy for at least 500 days. Transgenic platelets appeared normal with respect to surface antigens and response to platelet aggregation agonists. The highest-expressing transgenic line maintained human c-mpl ligand serum levels of 3 ng/mL. Megakaryocyte numbers in bone marrow and spleen were elevated, as were bone marrow and spleen megakaryocyte colony-forming cells (MEG-CFC). Megakaryocytes were observed in the bone marrow, spleen, liver, and lung, but in no other sites. Circulating myeloid and lymphoid cell populations were increased twofold. Additionally, the animals had a slight but significant anemia despite an increase in marrow colony-forming units-erythroid (CFU-E). No evidence of myelofibrosis was observed in the bone marrow. The platelet nadir in response to administration of either antiplatelet serum (APS) or 5-fluorouracil (5FU) was significantly reduced relative to the control level. Furthermore, the red blood cell (RBC) nadir was reduced relative to control levels in both models, suggesting that c-mpl ligand can directly or indirectly support the maintenance of erythrocyte levels following thrombopoietic insult.
Steel Factor Enhances Integrin-Mediated Tyrosine Phosphorylation of Focal Adhesion Kinase (pp125FAK) and Paxillin
Authors: Takahira Hiroyuki , Gotoh Akihiko , Ritchie Alec , Broxmeyer Hal E.
Published: October 2019

Integrin-mediated interaction of hematopoietic progenitor cells with bone marrow stromal extracellular matrix components is important in hematopoiesis. Focal adhesion kinase (pp125FAK) plays a central role in signal transduction through integrin receptors. We studied matrix-integrin interaction and subsequent signaling in human growth factor-dependent cell line, TF-1. Adherence of unstimulated TF-1 cells to fibronectin-coated wells was blocked by antiintegrin β1 and combination of anti-α4 with anti-α5 antibodies, indicating α4β1 and α5β1 integrin mediated adherence. Steel factor (SLF) increased TF-1 adhesion to fibronectin dose-dependently and 10−7 mol/L wortmannin suppressed SLF-induced adhesion. Immunoprecipitation and immunoblotting with antiphosphotyrosine antibody showed that adherence of TF-1 cells to fibronectin without cytokine caused tyrosine phosphorylation of several proteins identified as pp125FAK and paxillin. SLF induced spreading of adherent TF-1 cells and enhanced tyrosine phosphorylation of pp125FAK and paxillin in a dose-dependent manner. Treatment with SLF without plating on fibronectin did not induce tyrosine phosphorylation of pp125FAK. Wortmannin, at 10−7 mol/L, completely abolished SLF-induced enhancement of pp125FAK tyrosine phosphorylation, while c-kit autophosphorylation was not affected. This suggests that increase of pp125FAK tyrosine phosphorylation was mediated through a wortmannin sensitive pathway, rather than by direct action on c-kit tyrosine kinase. Treatment of adherent TF-1 cells with RGDS peptide plus anti-α4 antibody also inhibited SLF-induced enhancement of pp125FAK tyrosine phosphorylation without detachment of TF-1 cells. These data suggest that SLF enhances integrin-fibronectin-dependent tyrosine phosphorylation of pp125FAK through activation of integrin (“inside-out” signaling) and following integrin occupancy. This establishes a novel linkage between c-kit/SLF pathway and integrin fibronectin signaling.
Activation of Human Plasminogen by Staphylokinase. Direct Evidence That Preformed Plasmin Is Necessary for Activation to Occur
Authors: Grella Davida K. , Castellino Francis J.
Published: October 2019

To directly determine whether the mechanism of activation of human plasminogen (HPg) by staphylokinase (Sak) required formation of an active complex of Sak and HPg, recombinant (r) variants of HPg were examined that allowed dissection of the steps involved in this activation. The rate of activation of wild-type (wt) r-HPg by equimolar levels of Sak was enhanced when small amounts of human plasmin (HPm) were included, suggesting that a Sak-HPm complex was a more effective plasminogen activator than a putative Sak-HPg complex. Incubation of equimolar Sak with a cleavage site resistant mutant of HPg (r-[R561 A]HPg) did not result in generation of amidolytic activity of the complex, in contrast to a similar experiment with streptokinase (SK) in place of Sak, where substantial amidolytic activity was generated. This result supplies evidence that an active complex of Sak and HPg does not form, as is the case with SK. Another mutant, r-[D646E]HPg, which, upon activation, would lead to a form of HPm defective in enzymatic activity, is also not converted to its two-chain form by Sak, but is converted to the inactive two-chain form by urokinase, a direct plasminogen activator, and by equimolar complexes of SK or Sak with wtr-HPm. This shows that the active site of HPm is the functional plasminogen activator entity in the Sak-HPm complex. These results show that the mechanism of activation of HPg by Sak proceeds in a distinctly different manner than the similar activation by SK. Although SK does not require the presence of HPm for this activation, a necessary condition for the activation by Sak is formation of a small amount of HPm generated via another activation pathway. These different mechanisms have significant implications in production of the fibrinolytic state by these two indirect bacterial plasminogen activators.
Inhibition of the Defense System Stimulating Interleukin-12 Interferon-γ Pathway During Critical Illness
Authors: Ertel Wolfgang , Keel Marius , Neidhardt Regula , Steckholzer Ursula , Kremer Jean-Pierre , Ungethuem Udo , Trentz Otmar
Published: October 2019

Interleukin-12 (IL-12) and interferon-γ (IFN-γ) exert protective effects during experimental endotoxemia through upregulation of cellular immunity and phagocytic functions. They are part of a positive regulatory feedback loop that enhances the production of the other. Because critically ill patients show a marked suppression of T-cell and macrophage functions with a high susceptibility to infection, potential defects in the immunity/inflammation upregulating IL-12 IFN-γ pathway were studied. As an ex vivo model of endotoxemia, lipopolysaccharide (LPS) stimulated whole blood from 25 critically ill patients and 12 healthy individuals was incubated with either recombinant human (rh) IL-12 or rhIFN-γ, respectively. IFN-γ dose-dependently (P < .05) increased the release of IL-12 p40 and p70 into LPS-stimulated whole blood from healthy humans without effect in whole blood from critically ill patients. RhIL-12 p70 enhanced (P < .05) the secretion of IFN-γ in controls, while it was ineffective in LPS-stimulated whole blood from critically ill patients. The observed inhibition of the IL-12 IFN-γ pathway is not specific to LPS, since Staphylococcus aureus Cowan strain I (SAC)-stimulated whole blood from critically ill patients showed similar suppression. The secretion of IL-12 and IFN-γ was less reduced in critically ill patients when using isolated cultures of adherent cells or lymphocytes. Although preculture of whole blood from healthy humans with IL-10, but not with IL-4, mimicked suppression of the IL-12 IFN-γ pathway similar to that observed during critical illness, the release of antiinflammatory reacting cytokines (IL-4, IL-10, transforming growth factor [TGF]-β1 ) was decreased into LPS-stimulated whole blood from critically ill patients. These results indicate at least two mechanisms responsible for dramatic disturbances of the IL-12 IFN-γ pathway during critical illness: (1) deactivation of IL-12 and IFN-γ producing leukocytes in vivo early after the primary insult, and (2) presence of serum suppressive factors different from IL-4, IL-10, or TGF-β1 . Because IL-12 and IFN-γ upregulate essential immune functions, the marked inhibition of IL-12 and IFN-γ release may be pivotal for high susceptibility of critically ill patients to infection.