bluuebunny/crossref_metadata_2025 huggingface dataset:
Localization of Receptors for Vasoactive Intestinal Peptide, Somatostatin, and Substance P in Distinct Compartments of Human Lymphoid Organs
Authors: Reubi Jean Claude , Horisberger Ursula , Kappeler Andreas , Laissue Jean A.
Published: October 2019

Regulatory peptides, such as vasoactive intestinal peptide (VIP), somatostatin (SS), or substance P (SP), are considered to play a role in immune regulation. To localize the targets of these peptides in the human immune system, their receptors have been evaluated with in vitro receptor autoradiography in lymph nodes, tonsils, appendix, Peyer's patches, spleen, and thymus. The three peptide receptors were detected in all lymphoid tissues tested, but, unexpectedly, usually in distinct compartments. In lymph nodes, palatine tonsils, vermiform appendix, and Peyer's patches, VIP receptors were found in the CD3 positive zone around lymphoid follicles; SS receptors in the germinal centers of secondary follicles; and SP receptors mainly in interfollicular blood vessels. In the spleen, VIP receptors were detected in periarterial lymphatic sheaths, SS receptors in the red pulp, and SP receptors in the central arteries. In the thymus, VIP receptors were present in cortex and medulla, SS receptors in the medulla, and SP receptors in blood vessels. For comparison, cholecystokinin (CCK)-A and -B receptors were not demonstrated in any of these tissues. These results suggest a strong compartmentalization of the three peptide receptors in human lymphoid tissues and represent the molecular basis for the understanding of a very complex and interactive mode of action of these peptides.
Interleukin-7 Influences the Development of Thymic Dendritic Cells
Authors: Varas Alberto , Vicente Angeles , Sacedón Rosa , Zapata Agustı́n G.
Published: October 2019

Interleukin-7 (IL-7) has been shown to be a critical factor in B and T lymphopoiesis, and to influence the differentiation of myeloid cell lineages. In the present study we extend these results demonstrating that IL-7 also plays an important role in the development of thymic dendritic cells (DC). The addition of IL-7 to rat fetal thymus organ cultures (FTOC) resulted in a drastic increase in the number of CD3−CD4−CD8− cells, which mostly expressed typical DC markers, including major histocompatibility complex class II, OX-62, CD11b, CD68, and CD54. These cells exhibited morphological and ultrastructural features of DC, and were potent stimulators of the allogeneic mixed leukocyte reaction. Although increased numbers of DC were continuously generated throughout the culture period in the presence of IL-7, they were not actively dividing, indicating that DC in IL-7–treated cultures did not arise by expansion of pre-existing cells. Reduced DC numbers obtained after the addition of neutralizing anti–IL-7 antibodies to mouse FTOC confirmed the relevance of endogenously produced IL-7 on thymic DC development. Furthermore, the addition of IL-7 to FTOC derived from severe combined immunodeficient mice also generated large numbers of DC in the absence of thymocyte maturation.
Relationship Between Cleaved L-Selectin Levels and the Outcome of Acute Myeloid Leukemia
Authors: Extermann M. , Bacchi M. , Monai N. , Fopp M. , Fey M. , Tichelli A. , Schapira M. , Spertini O.
Published: October 2019

High plasma levels of the shed form of L-selectin (sL-selectin) are frequently detectable in acute myeloid leukemia (AML). sL-selectin can inhibit blast cell adhesion to vascular endothelium and may thereby influence the phenotype of AML. In this study, we have investigated the relationship between sL-selectin levels and clinical presentation or disease outcome in 100 patients with AML. Fifty-eight patients were found to have sL-selectin levels ≥3.12 μg/mL (≥3 SD above the mean of healthy controls: “increased”). Patients with extramedullary disease such as lymphadenopathies, splenomegaly, hepatomegaly, and/or muco-cutaneous infiltration had significantly increased sL-selectin levels (P < .001). sL-selectin levels were significantly heterogeneous in the French-American-British subtypes (P = .0003). Patients with “normal” sL-selectin levels had higher probability of achieving complete remission (CR) than with “increased” levels: 81% versus 64%, respectively (P = .06). When adjusting for clinically relevant covariates predictive for CR (sex, age, Auer rods), “normal” sL-selectin levels were significantly associated with CR (odds ratio, 3.08; 95% confidence interval [CI], 1.10 to 8.58;P = .03). Moreover, patients with “increased” sL-selectin levels (≥3.12 μg/mL) had shorter event-free survival (EFS) (median 7.3 v 12 months, P = .008) and overall survival (median 1 v 2.05 years, P = .03) than patients with sL-selectin <3.12 μg/mL. Multivariate statistical analysis (adjusted for age and presence of Auer rods) indicated that sL-selectin was an independent prognostic factor for EFS (hazard ratio [HR], 1.96; 95% CI, 1.21 to 3.17, P = .006) and overall survival (HR, 1.80; 95% CI, 1.09 to 2.98; P = .02). Thus, plasma sL-selectin may be a useful prognostic marker in the evaluation of AML at diagnosis.

© 1998 by The American Society of Hematology.
Apolipoprotein E 4 Allele as a Genetic Risk Factor for Left Ventricular Failure in Homozygous β-Thalassemia
Authors: Economou-Petersen Effrosini , Aessopos Athanassios , Kladi Athina , Flevari Panagiota , Karabatsos Fotis , Fragodimitri Christina , Nicolaidis Peter , Vrettou Helen , Vassilopoulos Dimitris , Karagiorga-Lagana Markissia , Kremastinos Dimitrios Th. , Petersen Michael B.
Published: October 2019

In homozygous β-thalassemia, the organ damage is mainly attributed to excessive iron deposition through the formation of oxygen free radicals. Despite appropriate transfusion and chelation therapy and low ferritin levels, patients still develop organ failure, heart failure being the main cause of death. This study was designed to determine whether the decreased antioxidant activity of the apolipoprotein E (APOE) 4 allele could represent a genetic risk factor for the development of left ventricular failure (LVF) in β-thalassemia homozygotes. A total of 251 Greek β-thalassemia homozygotes were studied. Patients were divided in three groups: group A (n = 151) with no cardiac impairment, group C (n = 47) with LVF, and 53 patients with LV dilatation and normal LV systolic function constituted the group B. DNA was obtained from all patients, and the polymerase chain reaction was used to analyze the polymorphism at the APOE locus. The APOE allele frequencies were compared with those of a Greek control sample of 216 healthy blood donors. Patients with no cardiac impairment had an APOE 4 allele frequency (7.9%) not different from population controls (6.5%, P > .05), while patients with LVF had a significantly higher frequency of APOE 4 (12.8%) than the controls (P < .05, odds ratio = 2.11, 95% confidence interval 1.03 to 4.32). The APOE 4 allele may represent an important genetic risk factor for the development of organ damage in homozygous β-thalassemia.

© 1998 by The American Society of Hematology.
Structure-Function Analyses of Thrombomodulin by Gene-Targeting in Mice: The Cytoplasmic Domain Is Not Required for Normal Fetal Development
Authors: Conway Edward M. , Pollefeyt Saskia , Cornelissen Jan , DeBaere Inky , Steiner-Mosonyi Marta , Weitz Jeffrey I. , Weiler-Guettler Hartmut , Carmeliet Peter , Collen Désiré
Published: October 2019

Thrombomodulin (TM) is a widely expressed glycoprotein receptor that plays a physiologically important role in maintaining normal hemostatic balance postnatally. Inactivation of the TM gene in mice results in embryonic lethality without thrombosis, suggesting that structures of TM not recognized to be involved in coagulation might be critical for normal fetal development. Therefore, the in vivo role of the cytoplasmic domain of TM was studied by using homologous recombination in ES cells to create mice that lack this region of TM (TMcyt/cyt). Cross-breeding of F1 TMwt/cyt mice (1 wild-type and 1 mutant allele) resulted in more than 300 healthy offspring with a normal Mendelian inheritance pattern of 25.7% TMwt/wt, 46.6% TMwt/cyt, and 27.7% TMcyt/cyt mice, indicating that the tail of TM is not necessary for normal fetal development. Phenotypic analyses showed that the TMcyt/cyt mice responded identically to their wild-type littermates after procoagulant, proinflammatory, and skin wound challenges. Plasma levels of plasminogen, plasminogen activator inhibitor 1 (PAI-1), and 2-antiplasmin were unaltered, but plasmin:2-antiplasmin (PAP) levels were significantly lower in TMcyt/cyt mice than in TMwt/wt mice (0.46 ± 0.2 and 1.99 ± 0.1 ng/mL, respectively; P < .001). Tissue levels of TM antigen were also unaffected. However, functional levels of plasma TM in the TMcyt/cyt mice, as measured by thrombin-dependent activation of protein C, were significantly increased (P < .001). This supported the hypothesis that suppression in PAP levels may be due to augmented activation of thrombin-activatable fibrinolysis inhibitor (TAFI), with resultant inhibition of plasmin generation. In conclusion, these studies exclude the cytoplasmic domain of TM from playing a role in the early embryonic lethality of TM-null mice and support its function in regulating plasmin generation in plasma.
Differential Methotrexate Resistance in Childhood T- Versus Common/PreB-Acute Lymphoblastic Leukemia Can Be Measured by an In Situ Thymidylate Synthase Inhibition Assay, But Not by the MTT Assay
Authors: Rots Marianne G. , Pieters Rob , Kaspers Gert-Jan L. , van Zantwijk Christina H. , Noordhuis Paul , Mauritz Rob , Veerman Anjo J.P. , Jansen Gerrit , Peters Godefridus J.
Published: October 2019

Methotrexate (MTX) is not cytotoxic to patient-derived acute lymphoblastic leukemia (ALL) cells in total-cell-kill assays, such as the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, putatively due to the rescue effects of hypoxanthine and thymidine released from dying cells. This was mimicked by a diminished methotrexate (MTX) cytotoxicity for the cell lines HL60 and U937 in the presence of hypoxanthine, thymidine, or lysed ALL cells. However, enzymatic depletion or inhibition of nucleoside membrane transport did not result in MTX dose-dependent cytotoxicity in patient samples. Alternatively, a thymidylate synthase inhibition assay (TSIA), based on inhibition of the TS-catalyzed conversion of 3H-dUMP to dTMP and 3H2O, correlated with the MTT assay for antifolate sensitivity in four human leukemia cell lines with different modes of MTX resistance. For 86 ALL patient samples, TSI50 values after 21 hours exposure to MTX were not different between T- and c/preB-ALL (P = .46). After 3 hours incubation with MTX followed by an 18-hour drug-free period, T-ALL samples were 3.4-fold more resistant to MTX compared with c/preB-ALL samples (P = .001) reflecting the clinical differences in MTX sensitivity. TSI50 values correlated with MTX accumulation (r = −.58, P < .001). In conclusion, the TSIA, but not the MTT assay, can measure dose-response curves for MTX in patient-derived ALL cells and showed relative MTX resistance in T-ALL compared with c/preB-ALL.
Treatment of Refractory and Relapsed Acute Myelogenous Leukemia With Combination Chemotherapy Plus the Multidrug Resistance Modulator PSC 833 (Valspodar)
Authors: Advani Ranjana , Saba Hussain I. , Tallman Martin S. , Rowe Jacob M. , Wiernik Peter H. , Ramek Joseph , Dugan Kathleen , Lum Bert , Villena Jenny , Davis Eric , Paietta Elisabeth , Litchman Manuel , Sikic Branimir I. , Greenberg Peter L.
Published: October 2019

A potential mechanism of chemotherapy resistance in acute myeloid leukemia (AML) is the multidrug resistance (MDR-1) gene product P-glycoprotein (P-gp), which is often overexpressed in myeloblasts from refractory or relapsed AML. In a multicenter phase II clinical trial, 37 patients with these poor risk forms of AML were treated with PSC 833 (Valspodar; Novartis Pharmaceutical Corporation, East Hanover, NJ), a potent inhibitor of the MDR-1 efflux pump, plus mitoxantrone, etoposide, and cytarabine (PSC-MEC). Pharmacokinetic (PK) interactions of etoposide and mitoxantrone with PSC were anticipated, measured in comparison with historical controls without PSC, and showed a 57% decrease in etoposide clearance (P = .001) and a 1.8-fold longer beta half-life for mitoxantrone in plasma (P < .05). The doses of mitoxantrone and etoposide were substantially reduced to compensate for these interactions and clinical toxicity and in Cohort II were well tolerated at dose levels of 4 mg/m2mitoxantrone, 40 mg/m2 etoposide, and 1 g/m2 C daily for 5 days. Overall, postchemotherapy marrow hypoplasia was achieved in 33 patients. Twelve patients (32%) achieved complete remission, four achieved partial remission, and 21 failed therapy. The PK observations correlated with enhanced toxicity. The probability of an infectious early death was 36% (4 of 11) in patients with high PK parameters for either drug versus 5% (1 of 20) in those with lower PK parameters (P = .04). P-gp function was assessed in 19 patients using rhodamine-123 efflux and its inhibition by PSC. The median percentage of blasts expressing P-gp was increased (49%) for leukemic cells with PSC-inhibitable rhodamine efflux compared with 17% in cases lacking PSC-inhibitable efflux (P = .004). PSC-MEC was relatively well tolerated in these patients with poor-risk AML, and had encouraging antileukemic effects. The Eastern Cooperative Oncology Group is currently testing this regimen versus standard MEC chemotherapy in a phase III trial, E2995, in a similar patient population.
Inflammatory Cytokines and Vascular Endothelial Growth Factor Stimulate the Release of Soluble Tie Receptor From Human Endothelial Cells Via Metalloprotease Activation
Authors: Yabkowitz Rachel , Meyer Susanne , Black Tabitha , Elliott Gary , Merewether Lee Anne , Yamane Harvey K.
Published: October 2019

Activation of endothelial cells, important in processes such as angiogenesis, is regulated by cell surface receptors, including those in the tyrosine kinase (RTK) family. Receptor activity, in turn, can be modulated by phosphorylation, turnover, or proteolytic release of a soluble extracellular domain. Previously, we demonstrated that release of soluble tie-1 receptor from endothelial cells by phorbol myristate acetate (PMA) is mediated through protein kinase C and a Ca2+-dependent protease. In this study, the release of soluble tie-1 was shown to be stimulated by inflammatory cytokines and vascular endothelial growth factor (VEGF), but not by growth factors such as basic fibroblast growth factor (bFGF) or transforming growth factor  (TGF). Release of soluble tie by tumor necrosis factor  (TNF) or VEGF occurred within 10 minutes of stimulation and reached maximal levels within 60 minutes. Specificity was shown by fluorescence-activated cell sorting (FACS) analysis; endothelial cells exhibited a significant decrease in cell surface tie-1 expression in response to TNF, whereas expression of epidermal growth factor receptor (EGF-R) and CD31 was stable. In contrast, tie-1 expression on megakaryoblastic UT-7 cells was unaffected by PMA or TNF. Sequence analysis of the cleaved receptor indicated that tie-1 was proteolyzed at the E749/S750 peptide bond in the proximal transmembrane domain. Moreover, the hydroxamic acid derivative BB-24 demonstrated dose-dependent inhibition of cytokine-, PMA-, and VEGF-stimulated shedding, suggesting that the tie-1 protease was a metalloprotease. Protease activity in a tie-1 peptide cleavage assay was (1) associated with endothelial cell membranes, (2) specifically activated in TNF-treated cells, and (3) inhibited by BB-24. Additionally, proliferation of endothelial cells in response to VEGF, but not bFGF, was inhibited by BB-24, suggesting that the release of soluble tie-1 receptor plays a role in VEGF-mediated proliferation. This study demonstrated that the release of soluble tie-1 from endothelial cells is stimulated by inflammatory cytokines and VEGF through the activation of an endothelial membrane-associated metalloprotease.
Effects of Novel RAR- and RXR-Selective Retinoids on Myeloid Leukemic Proliferation and Differentiation In Vitro
Authors: Shiohara Masaaki , Dawson Marcia I. , Hobbs Peter D. , Sawai Nobukuni , Higuchi Tsukasa , Koike Kenichi , Komiyama Atsushi , Koeffler H. Phillip
Published: October 2019

Retinoids such as all-trans-retinoic acid (ATRA) and 9-cis-retinoic acid (9-cis-RA) have an important role in many aspects of proliferation and differentiation of hematopoietic cells. They exert their effects by binding to retinoic acid receptors (RARs) and/or retinoid X receptors (RXRs). We studied the effects of novel retinoids on proliferation and differentiation of HL-60 and NB4 myeloid leukemic cells, as well as acute promyelocytic leukemia (APL) cells from patients. RXR-selective SR11345 (Retinoid C) had little ability to inhibit the clonal growth and to induce the differentiation of either HL-60 or NB4 cells. However, SR11276 (Retinoid E), which activated both the RAR and RXR classes, and SR11278 (Retinoid D), which activated the RAR subtypes , β, and γ, could inhibit clonal growth of both cell types, as well as leukemic cells from APL patients. The combination of ATRA and either SR11276 or SR11278 additively inhibited APL cell proliferation. SR11302 (Retinoid A), with reported anti-AP–1 activity and no activation of RARs and RXR and SR11363 (Retinoid B), which selectively activated RARβ and γ, were inactive. The clonal proliferation of both HL-60 and NB4 cells that were pulse-exposed to 10-9 mol/L ATRA, SR11276, SR11278, or SR11345 for 3 days, washed, and plated in methylcellulose culture were inhibited by 0%, 51%, 21%, and 1% for HL-60 cells and 43%, 41%, 35%, and 1% for NB4, respectively, compared with nontreated control cells. When the HL-60 cells were pulse-exposed to 10-9 mol/L of either SR11278 or SR11276, plus 10-9 mol/L ATRA for 3 days, colony numbers were reduced by 46% and 64%, respectively. Induction of leukemic cell differentiation as determined by the nitroblue tetrazolium (NBT) assay showed that the combination of 10-7 mol/L of either SR11278 or SR11276 with 10-7 mol/L ATRA had additive effects on HL-60 cells, NB4 cells, and fresh APL cells. Induction of CD11b expression on both HL-60 and NB4 cells occurs during their differentiation. Expression of this antigen was synergistically augmented by the combination of either 10-7 to 10-8 mol/L SR11278 or 10-7to 10-9 mol/L SR11276 with 10-9 mol/L ATRA compared with either analog alone in HL-60 cells. Expression of the novel myeloid specific transcription factor C/EBPɛ was increased by SR11278 and SR11276 in both the HL-60 and NB4 cell lines. We conclude that retinoids or combination of retinoids with specificities for both RAR and RXR may markedly enhance the ability of ATRA to inhibit clonal growth and induce differentiation of HL-60 and NB4 leukemic cells. This occurs in the absence of continuous contact with retinoids.
Immunosurveillance of Alglucerase Enzyme Therapy for Gaucher Patients: Induction of Humoral Tolerance in Seroconverted Patients After Repeat Administration
Authors: Rosenberg Mireille , Kingma Wytske , Fitzpatrick Mary Anne , Richards Susan M.
Published: October 2019

Alglucerase, a macrophage-targeted enzyme replacement therapy for Gaucher disease, has been successfully used for several years to improve clinical symptoms and reverse disease progression. As part of an immunosurveillance program, 1,122 Gaucher patients were monitored for antibody response to glucocerebrosidase, the active component of alglucerase. Seroconversion was detected in 142 patients (12.8%) by enzyme-linked immunosorbent assay (ELISA) and confirmed by radioimmunoprecipitation. The majority (75%) of the seroconverted population had no detectable levels of circulating inhibitory antibody as assessed by in vitro inhibition of enzymatic activity of the therapeutic molecule. Of the remaining patients with putative inhibitory antibodies, the majority had only low levels of serum inhibitory activity, which was transient. A very small number of patients were identified as developing true neutralizing antibodies, as defined by the development of antibodies that impacted clinical efficacy. Many of the patient antibody responses were also diminished with time. Eighty-two of the 142 seroconverted patients have stopped producing antibody to the molecule and appear tolerized. The mean time for humoral tolerization was 28 months from initiation of therapy. Of 64 seroconverted patients followed for at least 30 months of therapy, the tolerization rate was 93%. These results show that although 12.8% of the patients on therapy developed antibodies to the molecule, 90% of these patients became tolerized over time.
Inheritance in Erythropoietic Protoporphyria: A Common Wild-Type Ferrochelatase Allelic Variant With Low Expression Accounts for Clinical Manifestation
Authors: Gouya Laurent , Puy Herve , Lamoril Jerôme , Da Silva Vasco , Grandchamp Bernard , Nordmann Yves , Deybach Jean-Charles
Published: October 2019

Erythropoietic protoporphyria (EPP) is a rare autosomal dominant disorder of heme biosynthesis characterized by partial decrease in ferrochelatase (FECH; EC 4.99.1.1) activity with protoporphyrin overproduction and consequent painful skin photosensitivity and rarely liver disease. EPP is normally inherited in an autosomal dominant pattern with low clinical penetrance; the many different mutations that have been identified are restricted to one FECH allele, with the other one being free of any mutations. However, clinical manifestations of dominant EPP cannot be simply a matter ofFECH haploinsufficiency, because patients have enzyme levels that are lower than the expected 50%. From RNA analysis in one family with dominant EPP, we recently suggested that clinical expression required coinheritance of a normal FECH allele with low expression and a mutant FECH allele. We now show that (1) coinheritance of a FECH gene defect and a wild-type low-expressed allele is generally involved in the clinical expression of EPP; (2) the low-expressed allelic variant was strongly associated with a partial 5′ haplotype [−251G IVS1−23T IVS2μsatA9] that may be ancestral and was present in an estimated 10% of a control group of Caucasian origin; and (3) haplotyping allows the absolute risk of developing the disease to be predicted for those inheriting FECH EPP mutations. EPP may thus be considered as an inherited disorder that does not strictly follow recessive or dominant rules. It may represent a model for phenotype modulation by mild variation in expression of the wild-type allele in autosomal dominant diseases.
High-Level Expression of Mpl in Platelets and Megakaryocytes Is Independent of Thrombopoietin
Authors: Cohen-Solal Karine , Vitrat Natacha , Titeux Monique , Vainchenker William , Wendling Françoise
Published: October 2019

Thrombopoietin (TPO) is a hematopoietic growth factor that regulates megakaryocytopoiesis and platelet production through binding to its receptor, Mpl, encoded by the c-mpl proto-oncogene. Circulating levels of TPO are regulated by receptor-mediated uptake and degradation. To better understand this mode of TPO regulation, we examined whether expression of Mpl was regulated by its ligand. Using RNase protection analysis, we found no differences in the levels ofc-mpl transcripts in megakaryocytes (MKs) produced in vitro either in the presence or absence of TPO and in platelets (PLTs) obtained from mice hyperstimulated in vivo by ectopic secretion of TPO. Similarly, Western blot analysis of MKs produced in the presence or absence of TPO showed no difference in Mpl levels. Levels of Mpl, GpIIb, or P-selectin were virtually identical in platelet lysates obtained from normal, TPO knockout and mildly TPO-stimulated mice. In contrast, the expression of Mpl was significantly reduced in PLTs from severely thrombocythemic mice. These results show that TPO does not have a major effect on the transcription or translation of Mpl. However, they do suggest that an excess of circulating TPO can lead to the disappearance of Mpl from PLTs via catabolism.
Potential Role for Hyaluronan and the Hyaluronan Receptor RHAMM in Mobilization and Trafficking of Hematopoietic Progenitor Cells
Authors: Pilarski Linda M. , Pruski Eva , Wizniak Juanita , Paine Darlene , Seeberger Karen , Mant Michael J. , Brown Christopher B. , Belch Andrew R.
Published: October 2019

Although the mechanism(s) underlying mobilization of hematopoietic progenitor cells (HPCs) is unknown, detachment from the bone marrow (BM) microenvironment and motility are likely to play a role. This work analyzes the motile behavior of HPCs and the receptors involved. CD34+45lo/medScatterlo/med HPCs from granulocyte colony-stimulating factor (G-CSF)–mobilized blood and mobilized BM were compared with steady-state BM for their ability to bind hyaluronan (HA), their expression of the HA receptors RHAMM and CD44, and their motogenic behavior. Although RHAMM and CD44 are expressed by mobilized blood HPCs, function blocking monoclonal antibodies (MoAbs) identified RHAMM as a major HA binding receptor, with a less consistent participation by CD44. Permeabilization of mobilized blood HPCs showed a pool of intracellular (ic) RHAMM and a smaller pool of icCD44. In contrast, steady-state BM HPCs have significantly larger pools of icRHAMM and icCD44. Also, in contrast to mobilized blood HPCs, for steady-state BM HPCs, MoAbs to RHAMM and CD44 act as agonists to upregulate HA binding. The comparison between mobilized and steady-state BM HPCs suggests that G-CSF mobilization is associated with depletion of intracellular stores of HA receptors and modulates HA receptor usage. To confirm that mobilization alters the HA receptor distribution and usage by HPCs, samples of BM were collected at the peak of G-CSF mobilization in parallel with mobilized blood samples. HA receptor distribution of mobilized BM HPCs was closely matched with mobilized blood HPCs and different from steady-state BM HPCs. Mobilized BM HPCs had lower pools of icHA receptors, similar to those of mobilized blood HPCs. Treatment of mobilized BM HPCs with anti-RHAMM MoAb decreased HA binding, in contrast to steady-state BM HPCs. Thus, G-CSF mobilization may stimulate an autocrine stimulatory loop for HPCs in which HA interacts with basal levels of RHAMM and/or CD44 to stimulate receptor recycling. Consistent with this, treatment of HPCs with azide, nystatin, or cytochalasin B increased HA binding, implicating an energy-dependent process involving lipid rafts and the cytoskeleton. Of the sorted HPCs, 66% were adherent and 27% were motile on fibronectin plus HA. HPC adherence was inhibited by MoAbs to β1 integrin and CD44, but not to RHAMM, whereas HPC motility was inhibited by MoAb to RHAMM and β1 integrin, but not to CD44. This finding suggests that RHAMM and CD44 play reciprocal roles in adhesion and motility by HPCs. The G-CSF–associated alterations in RHAMM distribution and the RHAMM-dependent motility of HPCs suggest a potential role for HA and RHAMM in trafficking of HPCs and the possible use of HA as a mobilizing agent in vivo.
Hematopoietic Growth Factors Signal Through the Formation of Reactive Oxygen Species
Authors: Sattler Martin , Winkler Thomas , Verma Shalini , Byrne Christopher H. , Shrikhande Gautam , Salgia Ravi , Griffin James D.
Published: October 2019

Hematopoietic growth factors (HGFs) stimulate growth, differentiation, and prevent apoptosis of progenitor cells. Each growth factor has a specific cell surface receptor, which activates both unique and shared signal transduction pathways. We found that several HGFs, including granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), steel factor (SF), and thrombopoietin (TPO) induce a rapid increase in reactive oxygen species (ROS) in quiescent cells. In an effort to understand the potential biochemical and biological consequences of increased ROS in these cells, we exposed growth factor-deprived cells to hydrogen peroxide (H2O2) at concentrations that increased intracellular ROS. H2O2 induced a dose-dependent increase in tyrosine phosphorylation, including increased tyrosine phosphorylation of the GM-CSF receptor beta chain (βc), STAT5, and other signaling proteins. H2O2 also induced expression of the early response gene c-FOS, and G1- to S-phase transition, but not S- to G2/M-phase transition of MO7e cells. The cell permeable antioxidant pyrrolidine dithiocarbamate (PDTC) decreased the intracellular levels of ROS and inhibited tyrosine phosphorylation induced by GM-CSF in MO7e cells, suggesting that ROS generation plays an important role in GM-CSF signaling. Consistent with this notion, PDTC and two other antioxidants, N-acetyl cysteine and 2-mercaptoethanol, reduced growth and viability of MO7e cells. These results suggest that generation of ROS in response to HGFs may contribute to downstream signaling events, especially those involving tyrosine phosphorylation.
The Critical Interaction of Glycoprotein (GP) Ibβ With GPIX—A Genetic Cause of Bernard-Soulier Syndrome
Authors: Kenny Dermot , Morateck Patricia A. , Gill Joan C. , Montgomery Robert R.
Published: October 2019

Bernard-Soulier syndrome is an uncommon bleeding disorder caused by a quantitative or qualitative defect in the platelet glycoprotein (GP)Ib/IX complex. The complex is composed of four subunits, GPIb, GPIbβ, GPIX, and GPV. Here we describe the molecular basis of a novel Bernard-Soulier syndrome variant in a patient in whom GPIb and GPIX were undetectable on the platelet surface. DNA sequence analysis showed normal sequence for GPIb, GPIX, and GPV. The GPIbβ gene has been mapped to the 22q11.2 region of chromosome 22 which was deleted from one chromosome of this patient. There was a single nucleotide deletion within the codon for Ala 80 in GPIbβ within the other allele. This mutation causes a translational frame shift that encodes for 86 altered amino acids and predicts a premature stop 15 amino acids short of the length of the wild-type protein. Transient coexpression of the mutant GPIbβ in 293T cells with wild-type GPIb and GPIX resulted in the surface expression of GPIb, but the absence of GPIX. Moreover, when a plasmid encoding the wild-type GPIbβ was transiently transfected into Chinese hamster ovary cells stably expressing GP, which retain the capacity to reexpress GPIX, there was a significant increase in the surface expression of GPIX. In contrast, when the mutant GPIbβ was transiently transfected into these cells, GPIX was not reexpressed on the plasma surface. Thus, a deletion of one copy of GPIbβ and a single nucleotide deletion in the codon for Ala 80 within the remaining GPIbβ allele causes the Bernard-Soulier phenotype through an interaction of GPIbβ with GPIX resulting in the absence of GPIb on the plasma membrane. The interaction of GPIbβ with GPIX is essential for the functional expression of GPIb.
Molecular Mechanisms of Zinc-Dependent Leukocyte Adhesion Involving the Urokinase Receptor and β2-Integrins
Authors: Chavakis Triantafyllos , May Andreas E. , Preissner Klaus T. , Kanse Sandip M.
Published: October 2019

The trace element Zinc (Zn2+) has been implicated as a mediator in host defense, yet the molecular basis for its extracellular functions remains obscure. Here, we demonstrate that Zn2+can induce the adhesion of myelomonocytic cells to the endothelium, as well as to the provisional matrix proteins vitronectin (VN) and fibrinogen (FBG), which are pivotal steps for the recruitment of leukocytes into inflamed/injured tissue. Physiologic concentrations of Zn2+ increased the urokinase receptor (uPAR)-mediated adhesion of myelomonocytic cells to VN, whereas other divalent cations had smaller effects. Zn2+-induced cell adhesion to VN was abolished by cation chelators such as 1-10-phenanthroline, as well as by plasminogen activator inhibitor-1 (PAI-1) and a monoclonal antibody (MoAb) against uPAR. These characteristics could be recapitulated with a uPAR-transfected cell line emphasizing the specificity of this receptor system for Zn2+-dependent cell adhesion. Like urokinase (uPA), Zn2+ increased the binding of radiolabeled VN to uPAR-expressing cells, as well as the interaction of VN with immobilized uPAR in an isolated system. Moreover, Zn2+ enhanced leukocytic cell adhesion to FBG and endothelial cell monolayers by activating β2-integrins. Instead of the direct β2-integrin activation through the divalent cation binding site, Zn2+-induced integrin activation was mediated via uPAR, a crucial regulator of this system. The present study uncovers for the first time Zn2+-mediated cell adhesion mechanisms that may play a crucial role in modulating leukocyte adhesion to vessel wall components.
Expression of Epstein-Barr Virus BamHI-A Rightward Transcripts in Latently Infected B Cells From Peripheral Blood
Authors: Chen Honglin , Smith Paul , Ambinder Richard F. , Hayward S. Diane
Published: October 2019

In addition to the Epstein-Barr virus (EBV) EBNA and LMP latency genes, there is a family of alternatively spliced BamHI-A rightward transcripts (BARTs). These latency transcripts are highly expressed in the EBV-associated malignancies nasopharyngeal carcinoma and Burkitt’s lymphoma, and are expressed at lower levels in latently EBV-infected B-cell lines. The contribution of the BARTs to EBV biology or pathogenesis is unknown. Resting B cells have recently been recognized as a reservoir for EBV persistence in the peripheral blood. In these cells, EBV gene expression is tightly restricted and the only viral gene known to be consistently expressed is LMP2A. We used cell sorting and reverse-transcriptase polymerase chain reaction (RT-PCR) to examine whether BARTs are expressed in the restricted form of in vivo latency. Our results demonstrated that RNAs with splicing diagnostic for transcripts containing the BART RPMS1 and BARFO open-reading frames (ORFs) were expressed in CD19+ but not in CD23+ B cells isolated from peripheral blood of healthy individuals. The product of the proximal RPMS1 ORF has not previously been characterized. The RPMS1 ORF was shown to encode a 15-kD protein that localized to the nucleus of transfected cells. Expression of the BARTs in peripheral blood B cells suggests that the proteins encoded by these transcripts are likely to be important for maintenance of in vivo latency.
Correlation of Bcl-2 Rearrangement With Clinical Characteristics and Outcome in Indolent Follicular Lymphoma
Authors: López-Guillermo Armando , Cabanillas Fernando , McDonnell Timothy I. , McLaughlin Peter , Smith Terry , Pugh William , Hagemeister Fredrick , Rodrı́guez M. Alma , Romaguera Jorge E. , Younes Anas , Sarris Andreas H. , Preti H. Alejandro , Lee Ming-Seng
Published: October 2019

The t(14;18) translocation, which involves the bcl-2oncogene, occurs in follicular lymphomas (FL) at two common sites: the major breakpoint region (MBR) and the minor cluster region (mcr). The biological and clinical significance of these breakpoints is unknown. The bcl-2 breakpoint site was determined in 247 previously untreated patients (49% men; median age 52 years) with indolent FL (155 grade I, 83 grade II, and 8 grade III) to correlate it with pretreatment characteristics, response, and outcome. The bcl-2 breakpoint site was determined by a polymerase chain reaction method of peripheral blood (all cases), bone marrows (149 cases), and fresh lymph node biopsy specimens (68 cases). The breakpoint site occurred at MBR in 175 cases (71%) and atmcr in 27 (11%). In 45 cases (18%), no breakpoint was detected (germline). No significant relationship was found between the rearrangements and the expression of BLC-2 and BAXproteins. Patients’ germline for MBR and mcr tended to present more frequently with stage IV disease and higher β2-microglobulin (β2M) levels, whereas mcr-rearranged patients presented more frequently with early stage and normal β2M. The complete response rate of germline patients was significantly lower than that of MBR and mcr patients. An estimated 3-year failure-free survival (FFS) for mcr, MBR, and germline cases was 95%, 76%, and 57%, respectively (P < .001). Thebcl-2 breakpoint site was independent of serum β2M and lactate dehydrogenase in its correlation with FFS. In conclusion, thebcl-2 rearrangement site is an important prognostic factor in indolent FL, useful to identify patients who may require different treatment.
A New Fusion Gene TPM3-ALK in Anaplastic Large Cell Lymphoma Created by a (1;2)(q25;p23) Translocation
Authors: Lamant Laurence , Dastugue Nicole , Pulford Karen , Delsol Georges , Mariamé Bernard
Published: October 2019

Anaplastic large cell lymphomas (ALCL) are frequently associated with the t(2;5)(p23;q35). This translocation fuses the nucleophosmin (NPM) gene at 5q35, which encodes a nucleolar protein involved in shuttling ribonucleoproteins from the cytoplasm to the nucleus, to the anaplastic lymphoma kinase (ALK) gene at 2p23, encoding a tyrosine kinase receptor. In this report, we describe a typical case of ALCL whose malignant cells exhibited a novel (1;2)(q25;p23) translocation. These cells expressed ALK protein, but, in contrast to t(2;5)-positive ALCL (which show cytoplasmic, nuclear, and nucleolar staining), labeling was restricted to the malignant cell cytoplasm. Using a polymerase chain reaction (PCR)-based technique to walk on chromosome 2 from the known ALK gene across the breakpoint, we showed that the gene involved at 1q25 is TPM3, encoding a nonmuscular tropomyosin. We subsequently identified, using reverse transcription-PCR analysis of cases showing similar ALK cytoplasm-restricted staining, fusion of the ALK andTPM3 genes in 2 other cases of ALCL. The TPM3 gene has been previously found in papillary thyroid carcinomas as a fusion partner with the TRK kinase gene. We showed that TPM3 is constitutively expressed in lymphoid cell lines, suggesting that, in these t(1;2)-bearing ALCL cases, the TPM3 gene contributes an active promoter for ALK expression. Activation of the ALK catalytic domain probably results from homodimerization of the hybrid protein TPM3-ALK, through the TPM3 protein-protein interaction domain. The present cases of ALCL associated with a novel t(1;2)(q25;p23) demonstrate that at least one fusion partner other than NPM can activate the intracytoplasmic domain of the ALK kinase.
Prevention of Transfusion-Associated Graft-Versus-Host Disease by Photochemical Treatment
Authors: Grass Joshua A. , Wafa Tamim , Reames Aaron , Wages David , Corash Laurence , Ferrara James L.M. , Lin Lily
Published: October 2019

Photochemical treatment (PCT) with the psoralen S-59 and long wavelength ultraviolet light (UVA) inactivates high titers of contaminating viruses, bacteria, and leukocytes in human platelet concentrates. The present study evaluated the efficacy of PCT to prevent transfusion-associated graft-versus-host disease (TA-GVHD) in vivo using a well-characterized parent to F1 murine transfusion model. Recipient mice in four treatment groups were transfused with 108 splenic leukocytes. (1) Control group mice received syngeneic splenic leukocyte transfusions; (2) GVHD group mice received untreated allogeneic splenic leukocytes; (3) gamma radiation group mice received gamma irradiated (2,500 cGy) allogeneic splenic leukocytes; and (4) PCT group mice received allogeneic splenic leukocytes treated with 150 μmol/L S-59 and 2.1 J/cm2UVA. Multiple biological and clinical parameters were used to monitor the development of TA-GVHD in recipient mice over a 10-week posttransfusion observation period: peripheral blood cell levels, spleen size, engraftment by donor T cells, thymic cellularity, clinical signs of TA-GVHD (weight loss, activity, posture, fur texture, skin integrity), and histologic lesions of liver, spleen, bone marrow, and skin. Mice in the control group remained healthy and free of detectable disease. Mice in the GVHD group developed clinical and histological lesions of TA-GVHD, including pancytopenia, marked splenomegaly, wasting, engraftment with donor derived T cells, and thymic hypoplasia. In contrast, mice transfused with splenic leukocytes treated with (2,500 cGy) gamma radiation or 150 μmol/L S-59 and 2.1 J/cm2 UVA remained healthy and did not develop detectable TA-GVHD. Using an in vitro T-cell proliferation assay, greater than 105.1 murine T cells were inactivated by PCT. Therefore, in addition to inactivating high levels of pathogenic viruses and bacteria in PC, these data indicate that PCT is an effective alternative to gamma irradiation for prevention of TA-GVHD.
Differentiation of the Mononuclear Phagocyte System During Mouse Embryogenesis: The Role of Transcription Factor PU.1
Authors: Lichanska Agnieszka M. , Browne Catherine M. , Henkel Gregory W. , Murphy Kathleen M. , Ostrowski Michael C. , McKercher Scott R. , Maki Richard A. , Hume David A.
Published: October 2019

During mouse embryogenesis, macrophage-like cells arise first in the yolk sac and are produced subsequently in the liver. The onset of liver hematopoiesis is associated with the transition from primitive to definitive erythrocyte production. This report addresses the hypothesis that a similar transition in phenotype occurs in myelopoiesis. We have used whole mount in situ hybridization to detect macrophage-specific genes expressed during mouse development. The mouse c-fms mRNA, encoding the receptor for macrophage colony-stimulating factor (CSF-1), was expressed on phagocytic cells in the yolk sac and throughout the embryo before the onset of liver hematopoiesis. Similar cells were detected using the mannose receptor, the complement receptor (CR3), or the Microphthalmia transcription factor (MITF) as mRNA markers. By contrast, other markers including the F4/80 antigen, the macrophage scavenger receptor, the S-100 proteins, S100A8 and S100A9, and the secretory product lysozyme appeared later in development and appeared restricted to only a subset of c-fms–positive cells. Two-color immunolabeling on disaggregated cells confirmed that CR3 and c-fmsproteins are expressed on the same cells. Among the genes appearing later in development was the macrophage-restricted transcription factor, PU.1, which has been shown to be required for normal adult myelopoiesis. Mice with null mutations in PU.1 had normal numbers of c-fms–positive phagocytes at 11.5dpc. PU.1(−/−) embryonic stem cells were able to give rise to macrophage-like cells after cultivation in vitro. The results support previous evidence that yolk sac–derived fetal phagocytes are functionally distinct from those arising in the liver and develop via a different pathway.
Complexes of Heparin and Platelet Factor 4 Specifically Stimulate T Cells From Patients With Heparin-Induced Thrombocytopenia/Thrombosis
Authors: Bacsi S. , De Palma R. , Visentin G.P. , Gorski J. , Aster R.H.
Published: October 2019

Heparin-induced thrombocytopenia with thrombosis (HITT) is associated with antibodies specific for complexes consisting of heparin and platelet factor 4 (PF4). Studies in individual patients with HITT have demonstrated immunoglobulin (Ig) class switching from IgM to the IgG or IgA isotypes. This transition is thought to require helper T cells, but no studies of the cellular or molecular basis of this process have yet been reported. To characterize T-cell involvement in HITT, peripheral blood mononuclear cells (PBMC) from two patients with classical HITT obtained shortly after the acute episode were restimulated with heparin:PF4 complexes, PF4 alone, heparin alone, and medium alone in the presence of autologous antigen-presenting cells (APC). Responding T cells were then examined using the technique of “spectratyping,” in which sequences encoding CDR3 domains of individual V beta (BV) families are amplified and separated by gel electrophoresis. After 14 days in culture with antigen (heparin:PF4 complexes), but not after culture with PF4, heparin, or medium alone, patient cells, but not cells from normal subjects, preferentially expressed T-cell receptor (TCR)-containing β chains of the BV 5.1 family. Nucleotide sequencing of BV 5.1 TCR CDR3 showed that each patient had a personal repertoire, but also shared a tetrapeptide motif (PGTG). These findings provide evidence that the humoral immune response associated with HITT is driven by helper T cells that presumably recognize peptides derived from PF4. Identification of a common β-chain CDR3 motif in responding T cells from each of two patients suggests that a limited number of helper TCRs may be used to mount an antibody response to heparin:PF4 complexes. TCR spectratyping appears to offer a new way to examine the molecular basis of pathologic immune responses and may be useful in further studies of HITT and other immune-mediated hematologic disorders.
Rapid Mobilization of Intracellularly Stored RANTES in Response to Interferon-γ in Human Eosinophils
Authors: Lacy Paige , Mahmudi-Azer Salahaddin , Bablitz Ben , Hagen Stacey C. , Velazquez Juan R. , Man S.F. Paul , Moqbel Redwan
Published: October 2019

The CC chemokine RANTES is synthesized, stored, and upregulated in response to interferon-γ (IFN-γ) in human peripheral blood eosinophils. In this report, we propose that RANTES is rapidly mobilized from eosinophil crystalloid granules during agonist-induced degranulation. We stimulated purified eosinophils (>99%) from atopic asthmatics with 500 U/mL IFN-γ to analyze the kinetics of mobilization and release of RANTES (0 to 240 minutes). We used subcellular fractionation, immunogold analysis, two-color confocal laser scanning microscopy (CLSM), and enzyme-linked immunosorbent assay (ELISA) to trace the movement of eosinophil-derived RANTES from intracellular stores to release. RANTES was rapidly mobilized (10 minutes) and released after 120 minutes of stimulation (80 ± 15 pg/mL per 2 × 106 cells). RANTES appeared to be stored in at least two intracellular compartments: the matrix of crystalloid granules, detected by major basic protein and eosinophil peroxidase activities, and a specialized small secretory vesicle present in light membrane fractions. The extragranular RANTES was mobilized more rapidly than that of crystalloid granules during IFN-γ stimulation. This effect was not observed in eosinophils treated with IFN-, interleukin-3 (IL-3), IL-5, granulocyte-macrophage colony-stimulating factor (GM-CSF), or genistein followed by IFN-γ. Our findings suggest that RANTES may be mobilized and released by piecemeal degranulation upon stimulation, involving transport through a putative pool of small secretory vesicles.
The Importance of Antibody-Specificity in Determining Successful Radioimmunotherapy of B-Cell Lymphoma
Authors: Illidge Timothy M. , Cragg Mark S. , McBride Harry M. , French Ruth R. , Glennie Martin J.
Published: October 2019

We report the radioimmunotherapy of mouse B-cell lymphoma, BCL1, using a panel of anti–B-cell monoclonal antibodies (MoAb) (anti-CD19, anti-CD22, anti-major histocompatibility complex (MHC) II, and anti-idiotype (Id) radiolabeled with 131-iodine. When administered early in disease (day 4), the 131I–anti-MHCII MoAb cured tumors as a result of targeted irradiation alone, the unlabeled MoAb being nontherapeutic. In contrast,131I–anti-Id, despite targeting irradiation and having therapeutic activity as an unconjugated antibody, protected mice for only 30 days; 131I–anti-CD19 and anti-CD22 were therapeutically inactive. Binding and biodistribution studies showed that the anti-Id, unlike anti-MHCII, MoAb was cleared from target cells in vivo and delivered 4 times less irradiation to splenic tumor. Treating later in the disease (day 14) increased tumor load and produced the expected reduction in therapeutic activity with the anti-MHCII, but surprisingly, allowed 131I–anti-Id to cure most mice. This unexpected potency of 131I–anti-Id late in the disease appeared to result from the direct cytotoxicity of the anti-Id MoAb, which was more active in established disease, in combination with targeted irradiation. We believe the ability of targeted irradiation and certain cytotoxic MoAb to work cooperatively against tumor in this way has important implications for the selection of reagents in radioimmunotherapy of B-cell lymphoma.