bluuebunny/crossref_metadata_2025 huggingface dataset:
Long-Term Fetal Microchimerism in Peripheral Blood Mononuclear Cell Subsets in Healthy Women and Women With Scleroderma
Authors: Evans Paul C. , Lambert Nathalie , Maloney Sean , Furst Dan E. , Moore James M. , Nelson J. Lee
Published: October 2019

Fetal CD34+ CD38+ cells have recently been found to persist in maternal peripheral blood for many years after pregnancy. CD34+ CD38+ cells are progenitor cells that can differentiate into mature immune-competent cells. We asked whether long-term fetal microchimerism occurs in T lymphocyte, B lymphocyte, monocyte, and natural-killer cell populations of previously pregnant women. We targeted women with sons and used polymerase chain reaction for a Y-chromosome–specific sequence to test DNA extracted from peripheral blood mononuclear cells (PBMC) and from CD3, CD19, CD14, and CD56/16 sorted subsets. We also asked whether persistent microchimerism might contribute to subsequent autoimmune disease in the mother and included women with the autoimmune disease scleroderma. Scleroderma has a peak incidence in women after childbearing years and has clinical similarities to chronic graft-versus-host disease that occurs after allogeneic hematopoietic stem-cell transplantation, known to involve chimerism. Sixty-eight parous women were studied for male DNA in PBMC and 20 for PBMC subsets. Microchimerism was found in PBMC from 33% (16 of 48) of healthy women and 60% (12 of 20) women with scleroderma, P = .046. Microchimerism was found in some women in CD3, CD19, CD14, and CD56/16 subsets including up to 38 years after pregnancy. Microchimerism in PBMC subsets was not appreciably more frequent in scleroderma patients than in healthy controls. Overall, microchimerism was found in CD3, CD19, and CD14 subsets in approximately one third of women and in CD56/16 in one half of women. HLA typing of mothers and sons indicated that HLA compatibility was not a requirement for persistent microchimerism in PBMC subsets. Fetal microchimerism in the face of HLA disparity implies that specific maternal immunoregulatory pathways exist that permit persistence but prevent effector function of these cells in normal women. Although microchimerism in PBMC was more frequent in women with scleroderma than healthy controls additional studies will be necessary to determine whether microchimerism plays a role in the pathogenesis of this or other autoimmune diseases.
Mast Cell Migratory Response to Interleukin-8 Is Mediated Through Interaction With Chemokine Receptor CXCR2/Interleukin-8RB
Authors: Nilsson Gunnar , Mikovits Judy A. , Metcalfe Dean D. , Taub Dennis D.
Published: October 2019

To explore the role of chemokines in mast cell chemotaxis and accumulation at sites of inflammation, we first investigated the response of human mast cells to 18 different chemokines by induction of intracellular calcium mobilization in the human mast cell line, HMC-1. Only a subgroup of CXC chemokines defined by the conserved sequence motif glutamic acid-leucine-arginine (ELR) tripeptide motif, which included interleukin-8 (IL-8), growth-regulated oncogene  (GRO), neutrophil-activating peptide-2 (NAP-2), and epithelial cell–derived neutrophil activating peptide-78 (ENA-78), induced calcium flux in the cells. These observations suggested that the receptor CXCR2 (IL-8RB) should be expressed on the surface of these cells. Using the RNAse protection assay, CXCR2 mRNA, but not CXCR1 (IL-8RA) mRNA expression was detected in HMC-1 cells. Flow cytometry analysis documented the surface expression of CXCR2. A binding analysis performed with125I-IL-8 determined that there were approximately 3,600 high affinity IL-8 binding sites per HMC-1 cell, with a calculated kd of 1.2 to 2 nmol/L. The activity of this receptor was further explored using IL-8, which was found to induce dose-dependent chemotactic and haptotactic responses in both HMC-1 cells and in vitro cultured human cord blood–derived mast cells. These results show the expression of functional CXCR2 receptors on the surface of human mast cells, which may play an important role in mast cell recruitment during the genesis of an inflammatory response.
Recombinant Human Thrombopoietin in Combination With Granulocyte Colony-Stimulating Factor Enhances Mobilization of Peripheral Blood Progenitor Cells, Increases Peripheral Blood Platelet Concentration, and Accelerates Hematopoietic Recovery Following High-Dose Chemotherapy
Authors: Somlo George , Sniecinski Irena , ter Veer Anna , Longmate Jeffrey , Knutson Gaylord , Vuk-Pavlovic Stanimir , Bhatia Ravi , Chow Warren , Leong Lucille , Morgan Robert , Margolin Kim , Raschko James , Shibata Stephen , Tetef Merry , Yen Yun , Forman Stephen , Jones Dennie , Ashby Mark , Fyfe Gwen , Hellmann Susan , Doroshow James H.
Published: October 2019

Lineage-specific growth factors mobilize peripheral blood progenitor cells (PBPC) and accelerate hematopoietic recovery after high-dose chemotherapy. Recombinant human thrombopoietin (rhTPO) may further increase the progenitor-cell content and regenerating potential of PBPC products. We evaluated the safety and activity of rhTPO as a PBPC mobilizer in combination with granulocyte colony-stimulating factor (G-CSF) in 29 breast cancer patients treated with high-dose chemotherapy followed by PBPC reinfusion. Initially, patients received escalating single doses of rhTPO intravenously (IV) at 0.6, 1.2, or 2.4 μg/kg, on day 1. Subsequent patients received rhTPO 0.6 or 0.3 μg/kg on days −3, −1, and 1, or 0.6 μg/kg on days −1 and 1. G-CSF, 5 μg/kg IV or subcutaneously (SC) twice daily, was started on day 3 and continued through aphereses. Twenty comparable, concurrently and identically treated patients (who were eligible and would have been treated on protocol but for the lack of study opening) mobilized with G-CSF alone served as comparisons. CD34+ cell yields were substantially higher with the first apheresis following rhTPO and G-CSF versus G-CSF alone: 4.1 × 106/kg (range, 1.3 to 17.6) versus 0.8 × 106/ kg (range, 0.3 to 4.2), P = .0003. The targeted minimum yield of 3 × 106CD34+ cells/kg was procured following a single apheresis procedure in 61% of the rhTPO and G-CSF–mobilized group versus 10% of G-CSF–mobilized patients (P = .001). In rhTPO and G-CSF mobilized patients, granulocyte (day 8 v 9, P= .0001) and platelet recovery (day 9 v 10, P= .07) were accelerated, and fewer erythrocyte (3 v 4,P = .02) and platelet (4 v 5, P = .02) transfusions were needed compared with G-CSF–mobilized patients. Peripheral blood platelet counts, following rhTPO and G-CSF, were increased by greater than 100% and the platelet content of PBPC products by 60% to 110% on the first and second days of aphereses (P < .0001) with the greatest effect seen with repeated dosing of rhTPO at 0.6 μg/kg. rhTPO is safe and well tolerated as a mobilizing agent before PBPC collection. Mobilization with rhTPO and G-CSF, in comparison to a comparable, nonrandomized G-CSF–mobilized group of patients, decreases the number of apheresis procedures required, may accelerate hematopoietic recovery, and may reduce the number of transfusions required following high-dose chemotherapy for breast cancer.
Rapid Telomere Shortening in Children
Authors: Zeichner Steven L. , Palumbo Paul , Feng YanRu , Xiao Xiaodong , Gee Dennis , Sleasman John , Goodenow Maureen , Biggar Robert , Dimitrov Dimiter
Published: October 2019

Telomere shortening may reflect the total number of divisions experienced by a somatic cell and is associated with replicative senescence. We found that the average rate of telomere shortening in peripheral blood mononuclear cells (PBMCs) obtained longitudinally from nine different infants during the first 3 years of life (270 bp per year) is more than fourfold higher than in adults and does not correlate with telomerase activity. These results show that the rate of telomere loss changes during ontogeny, suggesting the existence of periods of accelerated cell division. Because human immunodeficiency virus (HIV) preferentially infects actively dividing cells, our observation suggesting accelerated cell division in children may provide an explanation for some of the distinctive pathogenic features of the HIV disease in infants, including higher viral loads and more rapid progression to acquired immunodeficiency syndrome (AIDS).
Commitment to the Monocytic Lineage Occurs in the Absence of the Transcription Factor PU.1
Authors: Henkel Gregory W. , McKercher Scott R. , Leenen Pieter J.M. , Maki Richard A.
Published: October 2019

Mice homozygous for the disruption of the PU.1 (Spi-1) gene do not produce mature macrophages. In determining the role of PU.1 in macrophage differentiation, the present study investigated whether or not there was commitment to the monocytic lineage in the absence of PU.1. Early PU.1−/− myeloid colonies were generated from neonate liver under conditions that promote primarily macrophage and granulocyte/macrophage colonies. These PU.1−/− colonies were found to contain cells with monocytic characteristics as determined by nonspecific esterase stain and the use of monoclonal antibodies that recognize early monocyte precursors, including Moma-2, ER-MP12, ER-MP20, and ER-MP58. In addition, early myeloid cells could be grown from PU.1−/− fetal liver cultures in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF). Similar to the PU.1 null colonies, the GM-CSF–dependent cells also possessed early monocytic characteristics, including the ability to phagocytize latex beads. The ability of PU.1−/− progenitors to commit to the monocytic lineage was also verified in vivo by flow cytometry and cytochemical analysis of primary neonate liver cells. The combined data shows that PU.1 is absolutely required for macrophage development after commitment to this lineage.
Vitamin E Reduces Monocyte Tissue Factor Expression in Cirrhotic Patients
Authors: Ferro Domenico , Basili Stefania , Praticó Domenico , Iuliano Luigi , FitzGerald Garret A. , Violi Francesco
Published: October 2019

Clotting activation may occur in liver cirrhosis, but the pathophysiological mechanism has not been fully elucidated. Because a previous study demonstrated that lipid peroxidation is increased in cirrhosis, we analyzed whether there is a relationship between lipid peroxidation and clotting activation. Thirty cirrhotic patients (19 men and 11 women; age, 34 to 79 years) and 30 controls matched for sex and age were investigated. In all subjects, monocyte expression of tissue factor (TF) antigen and activity; plasma levels of prothrombin fragment 1+2 (F1+2), a marker of thrombin generation; and urinary excretion of Isoprostane-F2-III, a marker of lipid peroxidation, were measured. Furthermore, the above-reported variables were re-evaluated after 30 days of treatment with standard therapy (n = 5) or standard therapy plus 300 mg vitamin E twice daily (n = 9). In addition, we analyzed in vitro if vitamin E (50 μmol/L) influenced monocyte TF expression and F1+2 generation. Cirrhotic patients had higher values of Isoprostane-F2-III (P < .0001), F1+2 (P < .0001), and monocyte TF antigen (P < .0001) and activity (P < .03) than controls. Isoprostane-F2-III was significantly correlated with F1+2 (Rho = 0.85; P < .0001) and TF antigen (Rho = 0.95; P < .0001) and activity (Rho = 0.94;P < .0001). After vitamin E treatment, Isoprostane-F2-III (P = .008), F1+2 (P < .008), and monocyte TF antigen (P = .012) and activity (P = .008) significantly decreased; no changes of these variables were detected in patients not receiving vitamin E. In vitro, vitamin E significantly reduced the expression of monocyte TF antigen (−52%; P = .001) and activity (−55%; P= .003), as well as F1+2 generation (−51%; P = .025). This study shows that vitamin E reduces both lipid peroxidation and clotting activation and suggests that lipid peroxidation may be an important mediator of clotting activation in liver cirrhosis.
Familial Pseudohyperkalemia Maps to the Same Locus as Dehydrated Hereditary Stomatocytosis (Hereditary Xerocytosis)
Authors: Iolascon A. , Stewart G.W. , Ajetunmobi J.F. , Perrotta S. , Delaunay J. , Carella M. , Zelante L. , Gasparini P.
Published: October 2019

Familial pseudohyperkalemia is a “leaky red blood cell” condition in which the cells show a temperature-dependent loss of potassium (K) from red blood cells when stored at room temperature, manifesting as apparent hyperkalemia. The red blood cells show a reduced lifespan in vivo but there is no frank hemolysis. Studies of cation content and transport show a marginal increase in permeability at 37°C and a degree of cellular dehydration, qualitatively similar to the changes seen in dehydrated hereditary stomatocytosis (hereditary xerocytosis). Physiological studies have shown that the passive leak to K has an abnormal temperature dependence, such that the leak is less sensitive to temperature than that in normal cells. We performed genetic mapping on the original family and found that the condition in this kindred maps to the same locus (16q23-ter) that we have previously identified for an Irish family with dehydrated hereditary stomatocytosis, which does not show the same temperature effects.
Kozak Sequence Polymorphism of the Glycoprotein (GP) Ib Gene Is a Major Determinant of the Plasma Membrane Levels of the Platelet GP Ib-IX-V Complex
Authors: Afshar-Kharghan Vahid , Li Chester Q. , Khoshnevis-Asl Mohammad , López José A.
Published: October 2019

Despite the known importance of the sequences surrounding ATG start codons (Kozak sequences) for efficient translation of proteins, few reports have appeared that describe the natural variations in these sequences. Here, we report a human polymorphism in the Kozak sequence of the platelet adhesion receptor, glycoprotein (GP) Ib, a component of the GP Ib-IX-V complex, which mediates the initial adhesion of platelets to the blood vessel wall following injury. The polymorphism is based on the presence of either thymine (T) or cytosine (C) at position −5 from the initiator ATG in the GP Ib gene. The less common allele, −5C, represented 8% to 17% of the alleles in four ethnic populations surveyed. This allele more closely resembles the sequence considered optimal for efficient initiation of protein translation and is associated with increased expression of the receptor on the cell membrane, both in transfected cells and in the platelets of individuals carrying the allele. In vitro transcription/translation studies indicate that the increased expression results from more efficient translation of the −5C form of the GP Ib mRNA. Other mutations made to approximate more closely the consensus sequence described by Kozak did not increase expression of the receptor. This is the first known description of Kozak sequence polymorphism as a determinant of the surface levels of a cell adhesion receptor. This polymorphism may influence an individual’s susceptibility for the development of cardiovascular disease.
Isochromosome 17q in Blast Crisis of Chronic Myeloid Leukemia and in Other Hematologic Malignancies Is the Result of Clustered Breakpoints in 17p11 and Is Not Associated With Coding TP53 Mutations
Authors: Fioretos Thoas , Strömbeck Bodil , Sandberg Therese , Johansson Bertil , Billström Rolf , Borg Åke , Nilsson Per-Gunnar , Van Den Berghe Herman , Hagemeijer Anne , Mitelman Felix , Höglund Mattias
Published: October 2019

An isochromosome of the long arm of chromosome 17, i(17q), is the most frequent genetic abnormality observed during the disease progression of Philadelphia chromosome–positive chronic myeloid leukemia (CML), and has been described as the sole anomaly in various other hematologic malignancies. The i(17q) hence plays a presumably important pathogenetic role both in leukemia development and progression. This notwithstanding, the molecular consequences of this abnormality have not been investigated in detail. We have analyzed 21 hematologic malignancies (8 CML in blast crisis, 8 myelodysplastic syndromes [MDS], 2 acute myeloid leukemias, 2 chronic lymphocytic leukemias, and 1 acute lymphoblastic leukemia) with i(17q) by fluorescence in situ hybridization (FISH). Using a yeast artificial chromosome (YAC) contig, derived from the short arm of chromosome 17, all cases were shown to have a breakpoint in 17p. In 12 cases, the breaks occurred within the Smith-Magenis Syndrome (SMS) common deletion region in 17p11, a gene-rich region which is genetically unstable. In 10 of these 12 cases, we were able to further map the breakpoints to specific markers localized within a single YAC clone. Six other cases showed breakpoints located proximally to the SMS common deletion region, but still within 17p11, and yet another case had a breakpoint distal to this region. Furthermore, using chromosome 17 centromere-specific probes, it could be shown that the majority of the i(17q) chromosomes (11 of 15 investigated cases) were dicentric, ie, they contained two centromeres, strongly suggesting that i(17q) is formed through an intrachromosomal recombination event, and also implicating that the i(17q), in a formal sense, should be designated idic(17)(p11). Because i(17q) formation results in loss of 17p material, potentially uncovering the effect of a tumor suppressor on the remaining 17p, the occurrence of TP53 mutations was studied in 17 cases by sequencing the entire coding region. In 16 cases, noTP53 mutations were found, whereas one MDS displayed a homozygous deletion of TP53. Thus, our data suggest that there is no association between i(17q) and coding TP53 mutations, and that another tumor suppressor gene(s), located in proximity of the SMS common deletion region, or in a more distal location, is of pathogenetic importance in i(17q)-associated leukemia.
Expression of the Human Immunodeficiency Virus-Tat Gene in Lymphoid Tissues of Transgenic Mice Is Associated With B-Cell Lymphoma
Authors: Kundu Ramendra K. , Sangiorgi Frank , Wu Lan-Ying , Pattengale Paul K. , Hinton David R. , Gill Parkash S. , Maxson Robert
Published: October 2019

The human immunodeficiency virus type 1 (HIV-1) Tat gene, a potent transactivator of viral and cellular genes, has been proposed as a key agent in the pathogenesis of acquired immune deficiency syndrome related disorders, including nonHodgkin’s lymphoma. In cultured cells, the HIV-1 Tat protein can induce the expression of the cytokines interleukin-6 (IL-6) and IL-10, which are known to induce proliferation and differentiation of lymphoid cells. Such alterations in cytokine expression, together with a secondary genetic event, are thought to ultimately lead to oncogenic transformation. To address the influence of Tat on lymphoid development in the context of the whole organism, we produced several transgenic mouse lines that express the Tat gene under the control of an actin promoter. We show here that this promoter directs expression to a variety of sites, including spleen, bone marrow, and lymph nodes. Approximately 25% to 30% of the Tat-transgenic population developed enlarged spleens within 1 year after birth. On histological examination, a significant number of spleens from Tat-transgenic mice exhibited malignant lymphoma of B-cell origin. IgG heavy chain rearrangement confirmed the clonal B-cell nature of these lymphoproliferations. In contrast, T-cell receptor genes exhibited a germline (unrearranged) structure. Reverse transcription polymerase chain reaction analysis of transgenic spleens revealed that mRNA encoding cytokines IL-6 and IL-10 was upregulated, suggesting a possible mechanism for the B-cell expansion in vivo.
Loss of Heterozygosity and Microsatellite Instability at theMLL Locus Are Common in Childhood Acute Leukemia, but not in Infant Acute Leukemia
Authors: Webb Julie C. , Golovleva Irina , Simpkins Alan H. , Kempski Helena , Reeves Brian , Sturt Natalie , Chessells Judith M. , Brickell Paul M.
Published: October 2019

Rearrangements involving the MLL gene at chromosome 11q23 are associated with leukemia and are present in up to 70% of infant leukemias. Loss of heterozygosity (LOH) has been shown for anonymous polymorphic markers at 11q23 in adult leukemias. To study LOH at theMLL locus, we have identified two new polymorphic microsatellite markers: a GAA repeat (mllGAAn) in intron 6 of theMLL gene and a GA (mllGAn) repeat in the 5′ flanking region of the gene, approximately 2 kb upstream of the translation initiation codon. The heterozygosity index of mllGAAn is 0.54, which renders it useful for analyzing LOH. We screened two groups of leukemia patients to study LOH at the mllGAAn marker. Group A (n = 18) was selected on the basis of presentation before 18 months. Cytogenetic and reverse transcription-polymerase chain reaction analysis showed that 9 of these 18 children had translocations involving MLL. No LOH was observed. Group B (n = 36) were randomly selected children who had presented with leukemia between 1993 and 1994. Cytogenetic analysis of this group showed a variety of different chromosomal abnormalities. LOH was shown in 9 of 20 individuals (45%) who were informative. Microsatellite instability (MSI) was demonstrated in 1 of 18 individuals in group A and 5 of 36 individuals (13.9%) in group B. MSI and LOH were observed simultaneously in three individuals. Loss of an allele was confirmed in one individual by fluorescence in situ hybridization. Individuals with MSI or LOH at mllGAAn were selected for analysis at anonymous polymorphic markers D11S1364 and D11S1356, which flank the MLL gene. No LOH or MSI was observed at these markers in those individuals who were informative. These results show that LOH at the MLL gene locus is a common event during leukemogenesis. Furthermore, the presence of MSI at this locus suggests that the region is a hotspot for genetic instability.
A Phase II Trial of 200% ProMACE-CytaBOM in Patients With Previously Untreated Aggressive Lymphomas: Analysis of Response, Toxicity, and Dose Intensity
Authors: Gordon Leo I. , Young Mary , Weller Edie , Habermann Thomas M. , Winter Jane N. , Glick John , Ghosh Chirantan , Flynn Patrick , Cassileth Peter A.
Published: October 2019

We showed in a phase I trial that the maximum tolerated dose of the ProMACE-CytaBOM regimen in patients with aggressive lymphoma was 200% (Gordon et al, J Clin Oncol 14:1275, 1996). Based on these observations, we initiated a phase II trial designed to determine response, toxicity, and dose intensity using this regimen. We analyzed 74 patients with advanced-stage (III or IV) or bulky stage II aggressive lymphoma. The overall complete response rate was 69% (72% in evaluable patients). With a median follow-up of 4.5 years, the median survival has not yet been reached. The 4-year survival rate is 73% (95% confidence interval [CI] 62, 83%) and no difference was observed among International Prognostic Index (IPI) groups. The 4-year disease-free survival was 71% (95% CI 58, 84%) with no statistical difference between patients with IPI 0 to 1 versus 2 to 4. The toxicity was acceptable, though the grade 4 hematologic toxicity rate for this regimen was 100%. Grade 4 nonhematologic toxicity was 36%. Three cases of either myelodysplastic syndrome or acute leukemia occurred at 7 months, 3.4 years, and 4.2 years after registration. Cytogenic analysis was available in two cases, showing inv(16) without French American British classification (FAB) M4 EO histology in one patient and a 5q-syndrome in the other. These data suggest that 200% ProMACE-CytaBOM with either granulocyte-macrophage colony-stimulating factor (GM-CSF) or G-CSF results in a high complete remission rate and a disease-free survival comparable to any prior risk-based analysis in aggressive lymphoma. Before using this regimen in general practice, phase III clinical trials should be conducted.
Studies on Treatment of Acute Promyelocytic Leukemia With Arsenic Trioxide: Remission Induction, Follow-Up, and Molecular Monitoring in 11 Newly Diagnosed and 47 Relapsed Acute Promyelocytic Leukemia Patients
Authors: Niu Chao , Yan Hua , Yu Ting , Sun Hui-Ping , Liu Jian-Xiang , Li Xiu-Song , Wu Wen , Zhang Fen-Qin , Chen Yu , Zhou Li , Li Jun-Min , Zeng Xiao-Ying , Yang Ren-Rong Ou , Yuan Mi-Man , Ren Mei-Yu , Gu Feng-Ying , Cao Qi , Gu Bo-Wei , Su Xin-Ying , Chen Guo-Qiang , Xiong Shu-Min , Zhang Ting-dong , Waxman Samuel , Wang Zhen-Yi , Chen Zhu , Hu Jiong , Shen Zhi-Xiang , Chen Sai-Juan
Published: October 2019

Fifty-eight acute promyelocytic leukemia (APL) patients (11 newly diagnosed and 47 relapsed) were studied for arsenic trioxide (As2O3) treatment. Clinical complete remission (CR) was obtained in 8 of 11 (72.7%) newly diagnosed cases. However, As2O3 treatment resulted in hepatic toxicity in 7 cases including 2 deaths, in contrast to the mild liver dysfunction in one third of the relapsed patients. Forty of forty-seven (85.1%) relapsed patients achieved CR. Two of three nonresponders showed clonal evolution at relapse, with disappearance of t(15;17) and PML-RAR fusion gene in 1 and shift to a dominant AML-1-ETO population in another, suggesting a correlation between PML-RAR expression and therapeutic response. In a follow-up of 33 relapsed cases over 7 to 48 months, the estimated disease-free survival (DFS) rates for 1 and 2 years were 63.6% and 41.6%, respectively, and the actual median DFS was 17 months. Patients with white blood cell (WBC) count below 10 × 109/L at relapse had better survival than those with WBC count over 10 × 109/L (P = .038). The duration of As2O3-induced CR was related to postremission therapy, because there was only 2 of 11 relapses in patients treated with As2O3 combined with chemotherapy, compared with 12 of 18 relapses with As2O3 alone (P = .01). Reverse transcription polymerase chain reaction (RT-PCR) analysis in both newly diagnosed and relapsed groups showed long-term use of As2O3 could lead to a molecular remission in some patients. We thus recommend that ATRA be used as first choice for remission induction in newly diagnosed APL cases, whereas As2O3 can be either used as a rescue for relapsed cases or included into multidrug consolidation/maintenance clinical trials.
Mutational Analysis in Murine Models for Myeloma-Associated Fanconi’s Syndrome or Cast Myeloma Nephropathy
Authors: Decourt C. , Rocca A. , Bridoux F. , Vrtovsnik F. , Preud’homme J.L. , Cogné M. , Touchard G.
Published: October 2019

We have designed an in vivo model in which murine hybridoma cell clones producing human Ig light chains (LC) are administred to mice. Depending on which monoclonal LC is expressed, this model mimicks either cast myeloma nephropathy or the pathological condition defined as myeloma-associated Fanconi’s syndrome (FS) with LC crystallization. Morphological alterations of the kidney cells are thus obtained in mice. All studied LC are closely related human monoclonal VκI proteins, which differ by a limited number of substitutions within the variable region. In the case of an FS monoclonal LC, we show that limited changes introduced through site-directed mutagenesis in the variable domain may suppress formation of intracellular crystals within tubular cells. We also show that multiple peculiarities of the variable region are simultaneously needed to allow LC crystallization; this property thus likely results from a unique LC tridimensional conformation imposed by concomitant somatic mutations of a specific germinally encoded framework.
The MAL Gene Is Expressed in Primary Mediastinal Large B-Cell Lymphoma
Authors: Copie-Bergman Christiane , Gaulard Philippe , Maouche-Chrétien Leı̈la , Brière Josette , Haioun Corinne , Alonso Miguel A. , Roméo Paul-Henri , Leroy Karen
Published: October 2019

Primary mediastinal large B-cell lymphoma (PMBL) appears to be a distinct clinicopathologic entity among diffuse large B-cell lymphomas (DLBLs). To find molecular alterations associated with this disease, we compared the mRNAs expressed in 3 PMBLs and 3 peripheral DLBLs by differential display-reverse transcription (DDRT) and identified a mRNA specifically expressed in PMBLs. Sequence analysis showed that this mRNA is encoded by the MAL gene, the expression of which was shown to be restricted to the T-cell lineage during hematopoiesis. MAL gene expression was demonstrated by Northern blot and reverse transcription-polymerase chain reaction (RT-PCR) in 8 of 12 PMBLs. However, there was little or no MAL gene expression in 8 peripheral DLBLs. Immunohistochemical analysis evidenced expression of MAL protein in tumoral B cells restricted to the PMBL subtype. Finally, Southern blot studies did not demonstrate rearrangement of the MAL gene. Altogether, our results indicate that MAL expression is recurrent in PMBLs, providing further evidence that PMBL represents a distinct entity among DLBLs. Because MAL protein is located in detergent-insoluble glycolipid-enriched membrane (GEM) domains involved in lymphocyte signal transduction, abnormal expression of MAL protein in the B-lymphoid lineage may have significant implications in PMBL lymphomagenesis.
The Proliferative Potential of Myeloma Plasma Cells Manifest in the SCID-hu Host
Authors: Yaccoby Shmuel , Epstein Joshua
Published: October 2019

The low proliferative activity of myeloma plasma cells prompted the notion that the clonotypic B cells that exist in the blood and bone marrow of all myeloma patients contain the proliferative myeloma cells (stem cell). We have exploited our severe combined immunodeficiency (SCID)-hu host system for primary myeloma to investigate whether myeloma plasma cells are capable of sustained proliferation. Purified CD38++CD45− plasma cells consistently grew and produced myeloma and its manifestations in SCID-hu hosts (8 of 9 experiments). In contrast, the plasma cell-depleted bone marrow cells from 6 patients did not grow or produce myeloma in SCID-hu hosts. Similarly, whereas plasma-cell containing blood cells from 4 patients grew and produced myeloma in hosts, neither the PC-depleted blood cells from 3 of the patients nor a blood specimen that did not contain plasma cells grew in SCID-hu hosts, regardless of their CD19-expressing cell contents. Also, in hosts injected with blood cells, although the myeloma cells were able to disseminate through the murine host system, they were only able to grow in the human bones within a human microenvironment and were not detectable in the murine blood or other organs. Interestingly, the circulating plasma cells appear to grow more avidly in the SCID-hu hosts than their bone marrow counterparts, suggesting that they represent a subpopulation of the plasma cells in the bone marrow. Although our studies clearly demonstrate the proliferative potential of myeloma plasma cells, they are suggestive, not conclusive, as to the existence of a preplasmacytic myeloma progenitor cell.
Human Immunodeficiency Virus Type 1 Protease Inhibitor Modulates Activation of Peripheral Blood CD4+ T Cells and Decreases Their Susceptibility to Apoptosis In Vitro and In Vivo
Authors: Sloand Elaine M. , Kumar Princy N. , Kim Sonnie , Chaudhuri Aniruddho , Weichold Frank F. , Young Neal S.
Published: October 2019

CD4+ T cells from patients with human immunodeficiency virus (HIV) infection undergo apoptosis at an increased rate, which leads to their depletion during disease progression. Both the Fas-Receptor (Fas-R) and interleukin-1β (IL-1β)–converting enzyme (ICE; caspase 1) appear to play a role in the mechanism of apoptosis of CD4+ lymphocytes. Although Fas-R is upregulated on both CD4+ and CD8+ cells in HIV-infected patients, results from our laboratory and others indicate that, in patients with advanced disease, CD4+ cells preferentially express ICE. Protease inhibitors have successfully halted the progression of HIV disease and increased CD4+ T counts. In this study, we examined the effect of protease inhibitors on Fas-R (CD95), ICE (caspase 1) expression, apoptosis, and cell death in CD4+ T cells of (1) HIV-infected patients who were receiving protease inhibitors, and (2) normal and patient CD4+ T cells cultured with a protease inhibitor in vitro. Fifteen patients with advanced HIV disease on treatment showed dramatically decreased CD4+ T-cell ICE expression, diminished apoptosis, and increased numbers of CD4+ cells within 6 weeks of institution of protease inhibitor therapy, and before down-modulation of Fas-R (CD95) expression was evident. To determine the role of HIV infection, we studied the effect of ritonavir, a protease inhibitor, on normal and patient cells in vitro. Stimulated and unstimulated normal CD4+ T cells, cultured with protease inhibitor, demonstrated markedly decreased apoptosis and ICE expression (P = .01). While Fas-R expression was not significantly altered during short-term culture by such treatment, Fas-Ligand (Fas-L) membrane expression of phytohemagglutinin (PHA)-stimulated blood lymphocytes was decreased by protease inhibitor. In the presence of ritonavir, CD4+ T cells from HIV-infected patients showed similar changes in ICE intracellular levels without alteration of Fas expression. In conclusion, protease inhibitors appear to decrease CD4+ T-cell ICE expression and apoptosis before they affect Fas-R expression in HIV-infected patients. This action was independent of HIV infection, as similar effects were seen in CD4+ T cells from normal controls. Some of the benefit of protease inhibitors may be related to modification of programmed cell death, which increases CD4+ T-cell number. Whether this is due to directly to the changes effected in the caspase system remains to be determined.
Simultaneous Activity of MRP1 and Pgp Is Correlated With In Vitro Resistance to Daunorubicin and With In Vivo Resistance in Adult Acute Myeloid Leukemia
Authors: Legrand Ollivier , Simonin Ghislaine , Beauchamp-Nicoud Anne , Zittoun Robert , Marie Jean-Pierre
Published: October 2019

In adult acute myeloid leukemia (AML), the weight of the contribution of the combined activity of Pgp and MRP1 to drug resistance is not known. To address this question, we compared the activity of these proteins to the in vitro resistance to daunorubicin (DNR), etoposide, and cytosine arabinoside (Ara-C), using the calcein-AM uptake and the 3-[4, 5-di-methyl-thiazol-2, 5-diphenyl] tetrazolium bromide (MTT) assay in 80 adult AML patients. We found no correlation or only a weak correlation between the in vitro drug resistance to DNR and etoposide and MRP1 or Pgp expression or function when tested separately. However, a strong correlation was observed between the simultaneous activity of MRP1 and Pgp (quantified as the modulation of calcein-AM uptake by cyclosporin A and probenecid) and the LC50 of DNR (r = .77, P < .0001). This emphasized the role of these two proteins, not separately, but together in the resistance to DNR. In contrast, Mvp/LRP expression did not correlate with the LC50 of DNR. A high level of simultaneous activity of Pgp and MRP1 was predictive of a poor treatment outcome (for achievement of CR [P = .008], duration of relapse-free survival [RFS; P = .01], and duration of overall survival [OS; P = .02]). In addition, high LC50 of DNR and high LC50 of etoposide together were also predictive of a poor treatment outcome (for duration of RFS [P= .02] and duration of OS [P = .02]). The unfavorable cytogenetic category was more closely associated with the combined activity of both MRP1 and Pgp (P = .002) than with the activity of Pgp or MRP1 separately. This could explain the poor prognosis and the in vitro resistance to daunorubicin in this group of patients. These data suggest that treatment outcome may be improved when cellular DNR and etoposide resistance can be circumvented or modulated. Modulation of not only Pgp but also MRP1 could be essential to attain this aim in adult AML.
Protracted and Variable Latency of Acute Lymphoblastic Leukemia AfterTEL-AML1 Gene Fusion In Utero
Authors: Wiemels Joseph L. , Ford Anthony M. , Van Wering Elisabeth R. , Postma Aleida , Greaves Mel
Published: October 2019

We report a pair of identical twins with concordant acute lymphoblastic leukemia (ALL). Unusually, their diagnoses were spaced 9 years apart at ages 5 and 14. Leukemic cells in both twins had aTEL-AML1 rearrangement, which was characterized at the DNA level by an adaptation of a long distance polymerase chain reaction (PCR) method. The genomic fusion sequence was identical in the two leukemias, indicative of a single cell origin in one fetus, in utero. At the time twin 1 was diagnosed (aged 5 years), the bone marrow of twin 2 was hematologically normal. However, retrospective scrutiny of the DNA from an archived slide with clonotypic TEL-AML1 primers showed that the presumptive preleukemic clone was present and disseminated 9 years before a clinical diagnosis. These data provide novel insight into the natural history of childhood leukemia and suggest that consequent to a prenatal initiation of a leukemic clone, most probably by TEL-AML fusion itself, the latency of ALL can be both extremely variable and protracted. This, in turn, is likely to reflect the timing of critical secondary events.
STAT5 Activation by BCR-Abl Contributes to Transformation of K562 Leukemia Cells
Authors: de Groot Rolf P. , Raaijmakers Jan A.M. , Lammers Jan-Willem J. , Jove Richard , Koenderman Leo
Published: October 2019

Signal transducers and activators of transcription (STATs) belong to a family of transcription factors that were originally identified as mediators of cytokine-induced gene expression. Recent evidence, however, has shown that certain members of the STAT family, including STAT3, are also involved in cellular transformation. Here we show that STAT5 also plays a role in cellular transformation by the BCR-Abl oncogene. In BCR-Abl transformed K562 cells, STAT5A and 5B are constitutively phosphorylated on tyrosine and are transcriptionally active. Moreover, expression of a dominant negative form of STAT5 shows that active STAT5 is necessary for the growth in soft agar of these cells. These results show that besides STAT3, STAT5 can also be involved in cellular transformation.
Serial Analysis of Gene Expression in Human Monocyte-Derived Dendritic Cells
Authors: Hashimoto Shin-ichi , Suzuki Takuji , Dong Hong-Yan , Nagai Shigenori , Yamazaki Nobuyuki , Matsushima Kouji
Published: October 2019

Dendritic cells (DCs) are professional antigen-presenting cells in the immune system and can be generated in vitro from hematopoietic progenitor cells in the bone marrow, CD34+ cord blood cells, precursor cells in the peripheral blood, and blood monocytes by culturing with granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-4, and tumor necrosis factor-. We have performed serial analysis of gene expression (SAGE) in DCs derived from human blood monocytes. A total of 58,540 tag sequences from a DC complementary DNA (cDNA) library represented more than 17,000 different genes, and these data were compared with SAGE analysis of tags from monocytes (Mo) and GM-CSF–induced macrophages (M◊). Many of the genes that were differentially expressed in DCs were identified as genes encoding proteins related to cell structure and cell motility. Interestingly, the highly expressed genes in DCs encode chemokines such as TARC, MDC, and MCP-4, which preferentially chemoattract Th2-type lymphocytes. Although DCs have been considered to be very heterogeneous, the identification of specific genes expressed in human Mo-derived DCs should provide candidate genes to define subsets of, the function of, and the maturation stage of DCs and possibly also to diagnose diseases in which DCs play a significant role, such as autoimmune diseases and neoplasms. This study represents the first extensive gene expression analysis in any type of DCs.
Requirement of Activation of JNK and p38 for Environmental Stress-Induced Erythroid Differentiation and Apoptosis and of Inhibition of ERK for Apoptosis
Authors: Nagata Yuka , Todokoro Kazuo
Published: October 2019

C-Jun amino terminal kinase/stress-activated protein kinases (JNK/SAPK) and p38 subgroups of mitogen-activated protein kinases have been suggested to play a critical role in apoptosis, cell growth, and/or differentiation. We found that a short exposure of SKT6 cells, which respond to erythropoietin (Epo) and induce erythroid differentiation, to osmotic or heat shock induced transient activation of JNK/SAPK and p38 and inactivation of ERK and resulted in erythroid differentiation without Epo, whereas long exposure of the cells to these stresses induced prolonged activation/inactivation of the same kinases and caused apoptosis. Inhibition of JNK/SAPK and p38 resulted in inhibition of stress-induced erythroid differentiation and apoptosis. Inhibition of ERK had no effect on stress-induced erythroid differentiation, but stimulated apoptosis. Activation of p38 and/or JNK/SAPK for a short time caused erythroid differentiation without Epo, although its prolonged activation induced apoptosis. Activation of ERK suppressed stress-induced apoptosis. These results indicate that short cellular stresses, inducing transient activation of JNK/SAPK and p38, lead to cell differentiation rather than apoptosis. Furthermore, activation of JNK/SAPK and p38 is required for both cell differentiation and apoptosis, and the duration of their activation may determine the cell fate, cell differentiation, and apoptosis. In contrast, inactivation of ERK is required for stress-induced apoptosis but not cell differentiation.
Loss of CCR2 Expression and Functional Response to Monocyte Chemotactic Protein (MCP-1) During the Differentiation of Human Monocytes: Role of Secreted MCP-1 in the Regulation of the Chemotactic Response
Authors: Fantuzzi Laura , Borghi Paola , Ciolli Veniero , Pavlakis George , Belardelli Filippo , Gessani Sandra
Published: October 2019

Human peripheral blood monocytes differentiate into macrophages when cultured in vitro for a few days. In the present study, we investigated the expression of C-C chemokine and CXCR4 receptors in monocytes at different stages of differentiation. Culturing of monocytes for 7 days resulted in a progressive decrease of the mRNA that encodes for CCR2 and CCR3, whereas the expression of mRNA for other chemokine receptors (CCR1, CCR4, CCR5, and CXCR4) was not substantially affected. The loss of CCR2 mRNA expression in 7-day–cultured macrophages was associated with a strong reduction in the receptor expression at the plasma membrane, as well as in the monocyte chemotactic protein (MCP-1) binding, as compared with freshly isolated monocytes. Furthermore, the biologic response to MCP-1, as measured by intracellular calcium ions increase and chemotactic response, was lost in 7-day–cultured macrophages. Differentiation of monocytes into macrophages also resulted in an increased secretion of MCP-1 that, at least in part, was responsible for the downmodulation of its receptor (CCR2). The loss of CCR2 expression and the parallel increase of MCP-1 secretion triggered by differentiation may represent a feedback mechanism in the regulation of the chemotactic response of monocytes/macrophages.
Homocysteine-Induced Endoplasmic Reticulum Stress and Growth Arrest Leads to Specific Changes in Gene Expression in Human Vascular Endothelial Cells
Authors: Outinen P. Andrew , Sood Sudesh K. , Pfeifer Sabine I. , Pamidi Sushmita , Podor Thomas J. , Li Jun , Weitz Jeffrey I. , Austin Richard C.
Published: October 2019

Alterations in the cellular redox potential by homocysteine promote endothelial cell (EC) dysfunction, an early event in the progression of atherothrombotic disease. In this study, we demonstrate that homocysteine causes endoplasmic reticulum (ER) stress and growth arrest in human umbilical vein endothelial cells (HUVEC). To determine if these effects reflect specific changes in gene expression, cDNA microarrays were screened using radiolabeled cDNA probes generated from mRNA derived from HUVEC, cultured in the absence or presence of homocysteine. Good correlation was observed between expression profiles determined by this method and by Northern blotting. Consistent with its adverse effects on the ER, homocysteine alters the expression of genes sensitive to ER stress (ie, GADD45, GADD153, ATF-4, YY1). Several other genes observed to be differentially expressed by homocysteine are known to mediate cell growth and differentiation (ie, GADD45, GADD153, Id-1, cyclin D1, FRA-2), a finding that supports the observation that homocysteine causes a dose-dependent decrease in DNA synthesis in HUVEC. Additional gene profiles also show that homocysteine decreases cellular antioxidant potential (glutathione peroxidase, NKEF-B PAG, superoxide dismutase, clusterin), which could potentially enhance the cytotoxic effects of agents or conditions known to cause oxidative damage. These results successfully demonstrate the use of cDNA microarrays in identifying homocysteine-respondent genes and indicate that homocysteine-induced ER stress and growth arrest reflect specific changes in gene expression in human vascular EC.
Cyclophilin B Binding to Platelets Supports Calcium-Dependent Adhesion to Collagen
Authors: Allain Fabrice , Durieux Sandrine , Denys Agnès , Carpentier Mathieu , Spik Geneviève
Published: October 2019

We have recently reported that cyclophilin B (CyPB), a secreted cyclosporine-binding protein, could bind to T lymphocytes through interactions with two types of binding sites. The first ones, referred to as type I, involve interactions with the conserved domain of CyPB and promote the endocytosis of surface-bound ligand, while the second type of binding sites, termed type II, are represented by glycosaminoglycans (GAG). Here, we further investigated the interactions of CyPB with blood cell populations. In addition to lymphocytes, CyPB was found to interact mainly with platelets. The binding is specific, with a dissociation constant (kd) of 9 ± 3 nmol/L and the number of sites estimated at 960 ± 60 per cell. Platelet glycosaminoglycans are not required for the interactions, but the binding is dramatically reduced by active cyclosporine derivatives. We then analyzed the biologic effects of CyPB and found a significant increase in platelet adhesion to collagen. Concurrently, CyPB initiates a transmembranous influx of Ca2+ and induces the phosphorylation of the P-20 light chains of myosin. Taken together, the present results demonstrate for the first time that extracellular CyPB specifically interacts with platelets through a functional receptor related to the lymphocyte type I binding sites and might act by regulating the activity of a receptor-operated membrane Ca2+ channel.