bluuebunny/crossref_metadata_2025 huggingface dataset:
Inhibition of Human Breast Carcinoma Growth by a Soluble Recombinant Human CD40 Ligand
Authors: Hirano Akio , Longo Dan L. , Taub Dennis D. , Ferris Douglas K. , Young Lawrence S. , Eliopoulos Arisitides G. , Agathanggelou Angelo , Cullen Nicky , Macartney James , Fanslow William C. , Murphy William J.
Published: October 2019

CD40 is present on B cells, monocytes, dendritic cells, and endothelial cells, as well as a variety of neoplastic cell types, including carcinomas. CD40 stimulation by an antibody has previously been demonstrated to induce activation-induced cell death in aggressive histology human B-cell lymphoma cell lines. Therefore, we wanted to assess the effects of a recombinant soluble human CD40 ligand (srhCD40L) on human breast carcinoma cell lines. Human breast carcinoma cell lines were examined for CD40 expression by flow cytometry. CD40 expression could be detected on several human breast cancer cell lines and this could be augmented with interferon-γ. The cell lines were then incubated with a srhCD40L to assess effects on in vitro growth. srhCD40L significantly inhibited the proliferation of the CD40+ human breast cancer cell lines. This inhibition could also be augmented with interferon-γ. Viability was also affected and this was shown to be due to increased apoptosis of the cell lines in response to the ligand. Treatment of tumor-bearing mice was then performed to assess the in vivo efficacy of the ligand. Treatment of tumor-bearing SCID mice with the ligand resulted in significant increases in survival. Thus, CD40 stimulation by its ligand directly inhibits human breast carcinoma cells in vitro and in vivo. These results suggest that srhCD40L may be of clinical use to inhibit human breast carcinoma growth.
Impairment of Plasmodium falciparum Growth in Thalassemic Red Blood Cells: Further Evidence by Using Biotin Labeling and Flow Cytometry
Authors: Pattanapanyasat Kovit , Yongvanitchit Kosol , Tongtawe Pongsri , Tachavanich Kalaya , Wanachiwanawin Wanchai , Fucharoen Suthat , Walsh Douglas S.
Published: October 2019

Certain red blood cell (RBC) disorders, including thalassemia, have been associated with an innate protection against malaria infection. However, many in vitro correlative studies have been inconclusive. To better understand the relationship between human RBCs with thalassemia hemoglobinopathies and susceptibility to in vitro infection, we used an in vitro coculture system that involved biotin labeling and flow cytometry to study the ability of normal and variant RBC populations in supporting the growth of Plasmodium falciparum malaria parasites. Results showed that both normal and thalassemic RBCs were susceptible to P falciparum invasion, but the parasite multiplication rates were significantly reduced in the thalassemic RBC populations. The growth inhibition was especially marked in RBCs from -thalassemia patients (both -thalassemia1/-thalassemia2 and -thalassemia1 heterozygote). Our observations support the contention that thalassemia confers protection against malaria and may explain why it is more prevalent in malaria endemic areas.
Loss of FancC Function Results in Decreased Hematopoietic Stem Cell Repopulating Ability
Authors: Haneline Laura S. , Gobbett Troy A. , Ramani Rema , Carreau Madeleine , Buchwald Manuel , Yoder Mervin C. , Clapp D. Wade
Published: October 2019

Fanconi anemia (FA) is a complex genetic disorder characterized by progressive bone marrow (BM) aplasia, chromosomal instability, and acquisition of malignancies, particularly myeloid leukemia. We used a murine model containing a disruption of the murine homologue ofFANCC (FancC) to evaluate short- and long-term multilineage repopulating ability of FancC −/− cells in vivo. Competitive repopulation assays were conducted where “test”FancC −/− or FancC +/+ BM cells (expressing CD45.2) were cotransplanted with congenic competitor cells (expressing CD45.1) into irradiated mice. In two independent experiments, we determined that FancC −/− BM cells have a profound decrease in short-term, as well as long-term, multilineage repopulating ability. To determine quantitatively the relative production of progeny cells by each test cell population, we calculated test cell contribution to chimerism as compared with 1 × 105 competitor cells. We determined that FancC −/− cells have a 7-fold to 12-fold decrease in repopulating ability compared with FancC +/+cells. These data indicate that loss of FancC function results in reduced in vivo repopulating ability of pluripotential hematopoietic stem cells, which may play a role in the development of the BM failure in FA patients. This model system provides a powerful tool for evaluation of experimental therapeutics on hematopoietic stem cell function.
Erythropoietin– and Stem Cell Factor–Induced DNA Synthesis in Normal Human Erythroid Progenitor Cells Requires Activation of Protein Kinase C and Is Strongly Inhibited by Thrombin
Authors: Haslauer Michael , Baltensperger Kurt , Porzig Hartmut
Published: October 2019

Proliferation, differentiation, and survival of erythroid progenitor cells are mainly regulated by stem cell factor (SCF) and erythropoietin (Epo). Using normal human progenitors, we analyzed the role of Ca2+-sensitive protein kinase C (PKC) subtypes and of G-protein–coupled receptor ligands on growth factor–dependent DNA synthesis. We show that stimulation of DNA synthesis by the two growth factors requires activation of PKC. Inhibitors of Ca2+-activated PKC subtypes blocked the growth factor–induced 3H-thymidine incorporation. SCF and Epo caused no significant translocation of PKC into the membrane, but treatment of intact cells with either of the two cytokines resulted in enhanced activity of immunoprecipitated cytosolic PKC. Stimulation of PKC with the phorbol ester PMA mimicked the cytokine effect on DNA synthesis. Epo-, SCF-, and PMA-induced thymidine incorporation was potently inhibited by thrombin (half-maximal inhibition with 0.1 U/mL). This effect was mediated via the G-protein-coupled thrombin receptor and the Rho guanosine triphosphatase. Adenosine diphosphate caused a modest Ca2+-dependent stimulation of DNA synthesis in the absence of cytokines and specifically enhanced the effect of SCF. Cyclic 3′,5′-adenosine monophosphate exerted a selective inhibitory effect on Epo-stimulated thymidine incorporation. Our results define PKC as major intermediate effector of cytokine signaling and suggest a role for thrombin in controlling erythroid progenitor proliferation.
von Willebrand Factor Propeptide in Vascular Disorders: A Tool to Distinguish Between Acute and Chronic Endothelial Cell Perturbation
Authors: van Mourik Jan A. , Boertjes Ria , Huisveld Inge A. , Fijnvandraat Karin , Pajkrt Dasja , van Genderen Perry J.J. , Fijnheer Rob
Published: October 2019

Before de novo synthesized von Willebrand factor (vWF) leaves the endothelial cell, it undergoes endoproteolytic cleavage of its propeptide (vW antigen II). The processed vWF and propeptide are either released constitutively or, following activation of the endothelium, released through the regulated pathway. In a recent study (Borchiellini et al, Blood 88:2951, 1996), we showed that the half-life of mature vWF and of its propeptide differ fourfold to fivefold. We postulated that the molar ratio of the propeptide to mature vWF could serve as a tool to assess the extent of endothelial cell activation under physiologic and clinical conditions. To test this hypothesis, we measured mature vWF and propeptide in patients with documented acute and chronic vascular disease, including patients with thrombotic thrombocytopenic purpura (TTP), acute septicemia, and diabetes mellitus. These data were compared with experimental conditions in healthy subjects in which perturbation of the endothelium was simulated by physical exercise or by administration of 1-deamino-8-D-arginine vasopressin (DDAVP) or endotoxin. In all individuals of the latter study group, both vWF and propeptide levels were elevated during the acute phase of the experimentally induced vascular perturbation; at later time points after stimulation, only vWF levels remained elevated. In patients with sepsis and TTP, both vWF and propeptide were elevated several-fold. Thus, this pattern can readily be explained in terms of acute perturbation of the endothelium. In contrast, in patients with diabetes mellitus propeptide levels were only slightly elevated, whereas vWF levels were elevated twofold to threefold. This pattern is a typical feature of chronic, low-grade activation of the endothelium. These observations support our hypothesis that measurement of both propeptide and vWF levels allows to discriminate between chronic and acute phases of endothelial cell activation in vivo. Measurement of only vWF is less indicative in this respect.
Identification of a Defect in the Intracellular Trafficking of a Kell Blood Group Variant
Authors: Yazdanbakhsh Karina , Lee Soohee , Yu Qian , Reid Marion E.
Published: October 2019

Blood group polymorphisms have been used as tools to study the architecture of the red blood cell (RBC) membrane. Some blood group variants have reduced antigen expression at the cell surface. Understanding the underlying mechanism for this reduced expression can potentially provide structural information and help to elucidate protein trafficking pathways of membrane proteins. The Kp(a+) phenotype is a variant in the Kell blood group system that is associated with a single amino acid substitution (R281W) in the Kell glycoprotein and serologically associated with a weakened expression of other Kell system antigens by an unknown mechanism. We found by immunoblotting of RBCs that the weakening of Kell antigens in this variant is due to a reduced amount of total Kell glycoprotein at the cell surface rather than to the inaccessibility of the antigens to Kell antibodies. Using a heterologous expression system, we demonstrate that the Kpa mutation causes retention of most of the Kell glycoprotein in a pre-Golgi compartment due to differential processing, thereby suggesting aberrant transport of the Kell protein to the cell surface. Furthermore, we demonstrated that single nucleotide substitutions into the coding region of the common KEL allele, as predicted by the molecular genotyping studies, was sufficient to encode three clinically significant low incidence antigens. We found that two low incidence antigens can be expressed on a single Kell protein, thus showing that the historical failure to detect such a variant is not due to structural constraints in the Kell protein. These studies demonstrate the power of studying the molecular mechanisms of blood group variants for elucidating the intracellular transport pathways of membrane proteins and the requirements for cell surface expression.
Hereditary Spherocytosis and Elliptocytosis Erythrocytes Show a Normal Transbilayer Phospholipid Distribution
Authors: de Jong Kitty , Larkin Sandra K. , Eber Stefan , Franck Paul F.H. , Roelofsen Ben , Kuypers Frans A.
Published: October 2019

Phosphatidylserine (PS) asymmetry was determined in red blood cells from patients with hereditary spherocytosis and elliptocytosis. No PS-exposing subpopulations were detected using the very sensitive method with fluorescently labeled annexin V. Treatment withN-ethylmaleimide or adenosine triphosphate (ATP) depletion to inactivate the flipase did not lead to formation of PS-exposing subpopulations in these cells, but elevated intracellular calcium levels did lead to extensive scrambling of the PS asymmetry. Although interactions of the membrane skeleton with the phospholipid bilayer have been suggested to stabilize the asymmetric distribution of PS across the bilayer, our data show that red blood cells with a severely damaged membrane skeleton are able to preserve asymmetry, even under conditions in which restoration of the asymmetric distribution is excluded. Moreover, the loss of membrane asymmetry in these cells requires active scrambling involving high levels of intracellular calcium as in normal cells. Our data show that the severe disorder of the membrane skeleton found in these cells does not affect the activity of flipase or scramblase, indicating that these proteins are not regulated by, nor coupled to the membrane skeleton assembly, and that possible thrombotic events in spherocytosis patients are not likely associated with altered PS topology of the red blood cells.
Embryonic Hemoglobins Are Expressed in Definitive Cells
Authors: Luo H.Y. , Liang X.L. , Frye C. , Wonio M. , Hankins G.D.V. , Chui D.H.K. , Alter B.P.
Published: October 2019

Human embryonic ζ and ɛ globin chains are synthesized in yolk sac–derived primitive erythroid cells, and decrease rapidly during definitive erythropoiesis. Examination of ζ and ɛ globin expression at the cellular level using dual-color immunofluorescence staining with specific monoclonal antibodies showed that embryonic globin proteins are present in definitive erythroid cells. More than half of fetal erythrocytes were positive for ζ and ∼5% for ɛ globin. Approximately one third of newborn red blood cells were ζ-positive and less than 1% ɛ-positive. Adult erythrocytes did not have embryonic globins. Erythroblasts that developed in liquid cultures also contained embryonic globin in amounts which declined with ontogenic age, and the proportion of positive cells in vitro was less than in the comparable erythrocytes that developed in vivo. Thus, embryonic globin chains are synthesized in definitive erythroid cells and decrease with ontogeny. Modulation of embryonic globin gene expression is not solely due to a switch from primitive to definitive erythropoiesis.
Genetic Evidence for an Additional Factor Required for Erythropoietin-Induced Signal Transduction
Authors: Gaffen Sarah L. , Lai Stephen Y. , Longmore Gregory D. , Liu Kathleen D. , Goldsmith Mark A.
Published: October 2019

Erythropoietin (EPO) and its receptor (EPOR) are required for the development of mature erythrocytes. After binding of ligand, the EPOR activates a variety of signaling pathways that ultimately control cellular proliferation, survival, and specific gene expression. Although erythroid progenitors appear to be the principal EPO-responsive cell type in vivo due to the restricted expression of the EPOR, many growth factor–dependent cell lines expressing the EPOR can respond to EPO by activating many or all of these pathways. In the present study, we have identified a cellular context (the interleukin-2 [IL-2]–dependent HT-2 line) in which the EPO stimulation of the EPOR fails to support cellular proliferation, STAT-5 induction, or MAPK activation, despite efficient phosphorylation of the EPOR and JAK2 and inhibition of apoptosis after withdrawal of IL-2. Interestingly, when we fused HT-2 cells expressing the EPOR with Ba/F3 cells in a complementation assay, the resulting hybridomas proliferated and potently activated STAT-5 and MAPK in response to EPO. These data indicate that an unidentified cellular factor is needed to mediate signaling by the EPOR. Moreover, Ba/F3 cells apparently express this factor(s) and somatic fusions can, therefore, confer EPO-responsiveness to HT-2 cells that lack this factor.
Formation of the Antithrombin Heterodimer In Vivo and the Onset of Thrombosis
Authors: Zhou Aiwu , Huntington James A. , Carrell Robin W.
Published: October 2019

Antithrombin is shown to undergo a slow spontaneous conversion to its inactive latent conformation with readily discernible amounts present in plasma on incubation at 37°C for 72 hours. More rapid conversion occurs on incubation of isolated antithrombin at 41°C or 50°C, but the appearance on electrophoresis of free latent antithrombin is preceded by the formation, in reciprocal proportions, of a new slow band. This slow component is shown to be a heterodimer of active and latent antithrombin. It can be isolated as a single stable band either by incubation of antithrombin or by mixing equimolar proportions of active and latent antithrombin under the same conditions that give overnight crystallization of the active/latent antithrombin heterodimer. Similarly, equimolar addition of latent antithrombin to plasma results electrophoretically in a quantitative shift to the slower heterodimer mobility. Clinically, the presence of latent antithrombin is potentially deleterious, because its linkage to form the heterodimer results in inactivation of the otherwise normal molecule linked to the latent antithrombin. In the case of -antithrombin, because the dimer readily dissociates, there is only a 11% additive loss of activity, but with β-antithrombin the dimer appears more stable, with the additive loss of activity from the normal β component being 21%, increasing to 33% on stabilization of the dimer with heparin. This linked and selective loss of activity of β-antithrombin provides an explanation for the unexpected severity of thrombotic episodes in heterozygotes with conformationally unstable antithrombins.
Tissue Factor and Factor VIIa Receptor/Ligand Interactions Induce Proinflammatory Effects in Macrophages
Authors: Cunningham Malcolm A. , Romas Pauline , Hutchinson Paul , Holdsworth Stephen R. , Tipping Peter G.
Published: October 2019

The potential for tissue factor (TF) to enhance inflammation by factor VIIa-dependent induction of proinflammatory changes in macrophages was explored. Purified recombinant human factor VIIa enhanced reactive oxygen species production by human monocyte-derived macrophages expressing TF in vitro. This effect was dose- and time-dependent, ligand- and receptor-specific, and independent of other coagulation proteins. This receptor/ligand binding induced phospholipase C-dependent intracellular calcium fluxes. Transfection studies using a human monocyte-derived cell line (U937) demonstrated that an intact intracytoplasmic domain of TF is required for factor VIIa-induced intracellular calcium fluxes. The capacity of TF to enhance proinflammatory functions of rabbit peritoneal-elicited macrophages (production of reactive oxygen species and expression of major histocompatibility complex class II and cell adhesion molecules) was demonstrated in vivo by treatment with an anti-TF antibody. These data demonstrate that, in addition to its role in activation of coagulation, TF can directly augment macrophage activation. These effects are initiated by binding factor VIIa and are independent of other coagulation proteins. These studies provide the first demonstration of a direct proinflammatory role for TF acting as a cell-signaling receptor.
Intrathymic Restriction and Peripheral Expansion of the T-Cell Repertoire in Omenn Syndrome
Authors: Signorini Simona , Imberti Luisa , Pirovano Silvia , Villa Anna , Facchetti Fabio , Ungari Marco , Bozzi Fabio , Albertini Alberto , Ugazio Alberto G. , Vezzoni Paolo , Notarangelo Luigi D.
Published: October 2019

Mutations in the human RAG genes that impair, but do not abolish, recombination activity lead to Omenn syndrome, a severe primary immune deficiency that is associated with clinical and pathological features of graft-versus-host disease and oligoclonal expansion of activated, autologous T cells. We have analyzed the mechanisms accounting for peripheral oligoclonality of the T-cell repertoire. Predominance of few T-cell receptor clonotypes (both within TCRAB- and within TCRGD-expressing lymphocytes) is already detectable in the thymus and is further selected for in the periphery, with a different distribution of clonotypes in different tissues. These data indicate that oligoclonality of the T-cell repertoire in Omenn syndrome is due both to intrathymic restriction and to peripheral expansion. Moreover, the RAG genes defect that causes Omenn syndrome directly affects early stages of V(D)J recombination, but does not alter the process of double-strand-break DNA repair, including N and P nucleotide insertion.
The Interleukin-12–Mediated Pathway of Immune Events Is Dysfunctional in Human Immunodeficiency Virus–Infected Individuals
Authors: Marshall Jason D. , Chehimi Jihed , Gri Giorgia , Kostman Jay R. , Montaner Luis J. , Trinchieri Giorgio
Published: October 2019

Interleukin-12 (IL-12) is a potentially critical factor in the immune response against human immunodeficiency virus (HIV) because it is important for regulating proliferation and interferon-γ (IFN-γ) production by T cells and natural killer (NK) cells, antigen presentation and accessory cell function by macrophages and dendritic cells, and cytolytic activities of cytotoxic T-lymphocyte cells and NK cells, which are all functions known to be dysfunctional in patients with acquired immune deficiency syndrome. Peripheral blood mononuclear cells (PBMC) from HIV-infected patients have been previously shown to be deficient in the ability to produce IL-12 in response to the bacterial pathogen Staphylococcus aureus Cowan. In this study, impaired IL-12 production in cells from PBMC of HIV-infected patients compared with healthy donors was observed across a broad panel of stimuli derived from infectious pathogens with or without priming with cytokines such as IFN-γ and IL-4, which amplify the IL-12 induction signal. Analysis of p40 and p35 mRNA accumulation showed that reductions in both subunits contribute to the lower IL-12 secretion of cells from HIV-infected individuals. PBMC from HIV-infected donors also failed to upregulate the IL-12 receptor β2 chain (IL-12Rβ2) in response to mitogenic stimuli. The expression of the IL-12Rβ2 gene could, however, be restored by in vitro exposure to rIL-12. Thus, it is possible that a primary IL-12 defect may lead to secondary deficiencies in expression of the genes for IL-12Rβ2 and IFN-γ, thus amplifying immune deficiency during HIV infection.
Stable Mixed Hematopoietic Chimerism in Dog Leukocyte Antigen–Identical Littermate Dogs Given Lymph Node Irradiation Before and Pharmacologic Immunosuppression After Marrow Transplantation
Authors: Storb Rainer , Yu Cong , Barnett Todd , Wagner John L. , Deeg H. Joachim , Nash Richard A. , Kiem Hans-Peter , McSweeney Peter , Seidel Kristy , Georges George , Zaucha J. Maciej
Published: October 2019

Stable mixed donor/host hematopoietic chimerism can be accomplished in dog leukocyte antigen (DLA)-identical littermate dogs given sublethal (200 cGy) total-body irradiation (TBI) before and immunosuppression with mycophenolate mofetil (MMF) and cyclosporine (CSP) after transplant (Blood 89:3048, 1997). Studies were based on the hypothesis that drugs that prevent graft-versus-host disease (GVHD) after transplant also suppress host-versus-graft (HVG) reactions and thereby enhance engraftment. Here, we asked whether pretransplant TBI provided marrow space for the graft to home or caused host immunosuppression. To address the questions, recipients were given pretransplant irradiation to cervical, thoracic, and abdominal lymph nodes (except pelvis), DLA-identical littermate marrow grafts, and MMF/CSP posttransplant. Six dogs that received 450 cGy irradiation showed initial engraftment. Two rejected their grafts after 8 and 18 weeks, 1 died with GVHD and engraftment, and 3 are alive as mixed chimeras after 57 to 97 weeks. Four dogs given 200 cGy irradiation also showed initial engraftment, but rejected their grafts after 10 to 18 weeks. Mixed chimerism was present in nonirradiated marrow and lymph node spaces and involved granulocytes, T cells, and monocytes. While other explanations are possible, results seem consistent with the hypothesis that pretransplant radiation provides host immunosuppression, and grafts can create their own marrow space. These data set the stage for the development of novel transplant regimens that substitute immunosuppressive for cytotoxic agents.
Extensive Venous and Arterial Thrombosis Associated With an Inhibitor to Activated Protein C
Authors: Zivelin Ariella , Gitel Sanford , Griffin John H. , Xu Xiao , Fernandez Jose A. , Martinowitz Uri , Cohen Yael , Halkin Hillel , Seligsohn Uri , Inbal Aida
Published: October 2019

Activated protein C resistance (APCR) in the absence of alterations in the factor V gene has been observed during pregnancy, in patients on oral contraceptives, in the presence of antiphospholipid antibodies, and in patients with ischemic stroke. We report a 49-year-old woman with recurrent major venous and arterial thromboses who displayed pronounced APCR, yet no changes in the activated protein C (APC) cleavage sites of factor V. The APCR values determined by four different assays were similar to those obtained in plasma from a homozygote for factor V Q506. Addition of IgG isolated from the patient’s serum to normal plasma lowered the APCR ratio from 2.4 to 1.6. Incubation of patient’s IgG with normal APC resulted in a profound change in the mobility of APC in crossed immunoelectrophoresis. APC was also shown to bind to patient’s IgG immobilized on a protein A agarose column. Factor Va inactivation by APC was inhibited by patient’s IgG, but not by control IgG in the presence or absence of either phospholipids or protein S. These results provide evidence for the existence of an acquired antibody against APC in the patient’s plasma, which gave rise to the APCR phenotype and was probably responsible for the major thrombotic events. We suggest that acquired APCR due to anti-APC antibodies be considered a potential cause for severe venous and arterial thromboses.
Interleukin-12 Therapy of Cutaneous T-Cell Lymphoma Induces Lesion Regression and Cytotoxic T-Cell Responses
Authors: Rook Alain H. , Wood Gary S. , Yoo Elisa K. , Elenitsas Rosalie , Kao David M.F. , Sherman Matthew L. , Witmer William K. , Rockwell Kenneth A. , Shane Ryan B. , Lessin Stuart R. , Vonderheid Eric C.
Published: October 2019

Progression of cutaneous T-cell lymphoma (CTCL) is associated with profound defects in cell-mediated immunity and depressed production of cytokines, which support cell-mediated immunity. Because we have observed marked defects in interleukin-12 (IL-12) production in CTCL and because IL-12 is critical for antitumor cytotoxic T-cell responses, we initiated a phase I dose escalation trial with recombinant human IL-12 (rhIL-12) where patients received either 50, 100, or 300 ng/kg rhIL-12 twice weekly subcutaneously or intralesionally for up to 24 weeks. Ten patients were entered: 5 with extensive plaque, 3 with Sezary syndrome, and 2 with extensive tumors with large cell transformation. One patient with Sezary syndrome dropped out after 1 week for personal reasons. Subcutaneous dosing resulted in complete responses (CR) in 2 of 5 plaque and partial responses (PR) in 2 of 5 plaque, and 1 of 2 Sezary syndrome (overall response rate CR+PR 5 of 9, 56%). A minor response also occurred in 1 of 5 plaque patients. Intralesional dosing resulted in individual tumor regression in 2 of 2 patients. Biopsy of regressing lesions showed a significant decrease in the density of the infiltrate in all cases and complete resolution of the infiltrate among those with clinical lesion resolution. An increase in numbers of CD8-positive and/or TIA-1–positive T cells were observed on immunohistochemical analysis of skin biopsy specimens obtained from regressing skin lesions. Adverse effects of rhIL-12 on this regimen were minor and limited and included low-grade fever and headache. One patient discontinued rhIL-12 at week 6 because of depression. These results suggest that rhIL-12 may augment antitumor cytotoxic T-cell responses and may represent a potent and well-tolerated therapeutic agent for CTCL.
The Incidence of Idiopathic Thrombocytopenic Purpura in Adults Increases With Age
Authors: Frederiksen Henrik , Schmidt Kai
Published: October 2019

With the aim of determining the incidence of idiopathic thrombocytopenic purpura (ITP) in adults, we searched all adult ITP patients diagnosed from April 1, 1973 to December 31, 1995 in the County of Funen in Denmark. This county comprises 9% of the total Danish adult population. A total of 221 patients fulfilled the inclusion criteria, yielding an annual standardized incidence rate of 2.68 per 100,000. The median age of the patient population was 56 years, and the female to male ratio was 1.7. Changing the platelet count cut-off point from 100 × 109/L to 50 × 109/L changed the incidence rate to 2.25 per 100,000. Comparing patients less and more than 60 years old, the incidence rate more than doubled and the sex difference was eliminated in the older age group. These two age groups were almost identical regarding platelet count at diagnosis and number of asymptomatic cases. The incidence rate increased in the study period. This increase in particular involved asymptomatic patients and old males who were both symptomatic or not symptomatic. Including additional patients identified by a questionnaire study of the contribution from the primary care physicians and the practicing specialists in the second half of the study period, a reliable estimate of the annual ITP incidence in Danish adults, using a platelet concentration cut-off point of 50 × 109/L, is 3.2 per 100,000 persons.
Dysregulated Myelopoiesis in Mice Lacking Jak3
Authors: Grossman William J. , Verbsky James W. , Yang Liping , Berg Leslie J. , Fields Larry E. , Chaplin David D. , Ratner Lee
Published: October 2019

Jak3 is a cytoplasmic tyrosine kinase that associates with the common chain of the interleukin-2 (IL-2) receptor and is involved in the function of the receptors for IL-2, IL-4, IL-7, IL-9, and IL-15. Mice deficient in Jak3 have few T and B cells, and no natural killer cells. Herein we show that the myeloid lineages in these mice are also affected by the loss of Jak3. Mice lacking Jak3 exhibit splenomegaly by 4 months of age. Peripheral blood smears show an increase in the number of neutrophils and cells of the monocytic lineage. Flow cytometry of splenocytes and peripheral blood show a significant increase in FcγRII/III(FcγR)/Mac-1, FcγR/Gr-1, and FcγR/F4/80 double-positive cells in −/− and +/− mice compared to wild-type mice, consistent with an expansion of cells of the myeloid lineages. In addition, as the mice age, F4/80 and CD3 positive mononuclear cells infiltrate the kidneys, lungs, and liver of these mice. When Jak3−/− mice are crossed with a transgenic mouse expressing Jak3 in the T and NK cell compartments, the splenomegaly and myeloid expansion are accentuated. These data correlate with the constitutive activation of T cells in the periphery as the transgenic cells lose their expression of Jak3 with age. However, when Jak3−/− mice are crossed with RAG-1–deficient animals, no splenomegaly or myeloid expansion is apparent. These results indicate that the loss of Jak3 in the T-cell compartment drives the expansion of the myeloid lineages.
Hyaluronate-Enhanced Hematopoiesis: Two Different Receptors Trigger the Release of Interleukin-1β and Interleukin-6 From Bone Marrow Macrophages
Authors: Khaldoyanidi Sophia , Moll Jürgen , Karakhanova Svetlana , Herrlich Peter , Ponta Helmut
Published: October 2019

The glycosaminoglycan hyaluronate (HA) is part of the extracellular environment in bone marrow. We show here that HA activates signal transduction cascades important for hemopoiesis. In myeloid and lymphoid long-term bone marrow cultures (LTBMC), treatment with hyaluronidase (HA’ase) results in reduced production of both progenitor and mature cells. Exogeneous HA added to LTBMC had the opposite effect: it enhanced hematopoiesis. The effect of HA is mediated through two different HA receptors on bone marrow macrophage-like cells, one of which is CD44 while the other is unknown. HA induces bone marrow macrophages to secrete IL-1β (CD44-dependent) and IL-6 (CD44-independent). The two receptors address different signal transduction pathways: CD44 links to a pathway activating p38 protein kinase while the other yet unknown receptor induces Erk activity. There was no difference of the effect of HA and HA’ase on hematopoiesis in LTBMC and on cytokine production by macrophages in CD44-deficient mice compared with wild-type mice, indicating that the CD44 hyaluronate receptor and its signal transduction can be compensated for. Our data suggest a regulatory role for the extracellular matrix component HA in hematopoiesis and show the induction of signal transduction by HA receptors.
Sickle Cell Acute Chest Syndrome: Pathogenesis and Rationale for Treatment
Authors: Stuart Marie J. , Setty B.N. Yamaja
Published: October 2019

Acute chest syndrome (ACS) is a leading cause of death in sickle cell disease (SCD). Our previous work showed that hypoxia enhances the ability of sickle erythrocytes to adhere to human microvessel endothelium via interaction between very late activation antigen-4 (VLA4) expressed on sickle erythrocytes and the endothelial adhesion molecule vascular cell adhesion molecule-1 (VCAM-1). Additionally, hypoxia has been shown to decrease the production of nitric oxide (NO) which inhibits VCAM-1 upregulation. Based on these observations, we hypothesize that during ACS, the rapidly progressive clinical course that can occur is caused by initial hypoxia-induced pulmonary endothelial VCAM-1 upregulation that is not counterbalanced by production of cytoprotective mediators, including NO, resulting in intrapulmonary adhesion. We assessed plasma NO metabolites and soluble VCAM-1 in 36 patients with SCD and 23 age-matched controls. Patients with SCD were evaluated at baseline (n = 36), in vaso-occlusive crisis (VOC; n = 12), and during ACS (n = 7). We observed marked upregulation of VCAM-1 during ACS (1,290 ± 451 ng per mL; mean ± 1 SD) with values significantly higher than controls (P < .0001) or patients either in steady state or VOC (P < .01). NO metabolites were concomitantly decreased during ACS (9.2 ± 1.5 nmol/mL) with values lower than controls (22.2 ± 5.5), patients during steady state (21.4 ± 5.5), or VOC (14.2 ± 1.2) (P< .0001). Additionally, the ratio of soluble VCAM-1 to NO metabolites during ACS (132.9 ± 46.5) was significantly higher when compared with controls (P < .0001) or patients either in steady state or VOC (P < .0001). Although hypoxia enhanced in vitro sickle erythrocyte-pulmonary microvessel adhesion, NO donors inhibited this process with concomitant inhibition of VCAM-1. We suggest that in ACS there is pathologic over expression of endothelial VCAM-1. Our investigations also provide a rationale for the therapeutic use in ACS of cytoprotective modulators including NO and dexamethasone, which potentially exert their efficacy by an inhibitory effect on VCAM-1 and concomitant inhibition of sickle erythrocyte-endothelial adhesion.
A Phase I-II Clinical Trial to Evaluate Removal of CD4 Cells and Partial Depletion of CD8 Cells From Donor Marrow for HLA-Mismatched Unrelated Recipients
Authors: Martin Paul J. , Rowley Scott D. , Anasetti Claudio , Chauncey Thomas R. , Gooley Ted , Petersdorf Effie W. , van Burik Jo-Anne , Flowers Mary E.D. , Storb Rainer , Appelbaum Frederick R. , Hansen John A.
Published: October 2019

We conducted a phase I-II clinical trial to test the hypothesis that removal of CD4 cells from an HLA-mismatched unrelated marrow graft would substantially reduce the risk of grades III-IV graft-versus-host disease (GVHD) and that retention of a specified number of CD8 cells in the graft would be sufficient to prevent rejection. Patients were eligible for this study when an HLA-A, -B, or -DRB1–matched unrelated donor could not be identified. HLA matching of the donor and recipient was based on typing of HLA-A and -B antigens by serologic methods and by typing of HLA-DRB1 alleles by molecular methods, and donors were selected when disparity was limited to a single HLA-DRB1 allele or a single HLA-A or -B antigen. Twenty-seven patients with hematologic malignancy or aplastic anemia were prepared to receive a transplant with conventional regimens of cyclophosphamide and fractionated total body irradiation, and a standard regimen of methotrexate and cyclosporine was given for GVHD prophylaxis. CD4 cells were removed from the donor marrow, and the numbers of CD8 cells were adjusted systematically in graded steps for successive patients, depending on the occurrence of grades III-IV GVHD or graft failure in previously enrolled patients. Removal of CD4 cells did not cause graft rejection or appreciably decrease the risk of grades III-IV GVHD. Depletion of CD8 cells was associated with an increased risk of rejection with either HLA-DRB1 disparity or with HLA-A or -B disparity. With either type of disparity, the risk of grades III-IV GVHD is likely to be higher than 15% at any dose of CD8 cells associated with less than 5% risk of graft failure. The absence of graft failure associated with CD4 depletion supports the hypothesis that donor CD4 cells are not essential for preventing marrow graft rejection in humans. The correlation between graft failure and the number of CD8 cells in the donor marrow supports the hypothesis that donor CD8 cells help to prevent marrow graft rejection.
BCR/ABL mRNA and the P210BCR/ABL Protein Are Downmodulated by Interferon- in Chronic Myeloid Leukemia Patients
Authors: Pane Fabrizio , Mostarda Ilaria , Selleri Carmine , Salzano Rossella , Raiola Anna Maria , Luciano Luigia , Saglio Giuseppe , Rotoli Bruno , Salvatore Francesco
Published: October 2019

The BCR/ABL hybrid gene plays a central role in the pathogenesis of the chronic phase of chronic myeloid leukemia (CML). We used a very sensitive quantitative reverse transcriptase-polymerase chain reaction to investigate the levels of hybrid BCR/ABL mRNA in bone marrow cells of 20 patients with Philadelphia positive (Ph+) CML treated with interferon- (IFN-) as a single agent. Bone marrow samples were collected at diagnosis and at hematologic remission induced by IFN-, or by hydroxyurea in case of resistance to IFN-. The mean levels of BCR/ABL transcripts in bone marrow mononuclear cells of patients who showed a complete hematologic response to IFN- were significantly reduced with respect to those at diagnosis (48 × 103v168 × 103; P < .001), whereas no difference was detected between the values at diagnosis and at hematologic remission in patients resistant to IFN-. In cell culture experiments, IFN- priming significantly reduced the levels of BCR/ABL hybrid transcripts in a dose-dependent manner in Ph+ bone marrow precursors obtained at diagnosis from patients who subsequently responded to IFN- treatment (P < .005). No downmodulation was observed in bone marrow precursors from patients who subsequently proved to be IFN-resistant. These results indicate that downmodulation of BCR/ABL gene expression could be one of the mechanisms involved in the response of CML patients to IFN- treatment.
Rapid Induction of CD40 on a Subset of Granulocyte Colony-Stimulating Factor–Mobilized CD34+ Blood Cells Identifies Myeloid Committed Progenitors and Permits Selection of Nonimmunogenic CD40− Progenitor Cells
Authors: Rondelli Damiano , Lemoli Roberto M. , Ratta Marina , Fogli Miriam , Re Francesca , Curti Antonio , Arpinati Mario , Tura Sante
Published: October 2019

CD40 antigen is a costimulatory molecule highly expressed on dendritic cells (DC) and activated B cells, which induces T-cell proliferation through the binding with CD40L receptor. In this study, we evaluated CD40 expression on normal CD34+blood cells and functionally characterized CD34+CD40+ and CD34+CD40− cell subsets. CD40, CD80, and CD86 antigens were constitutively expressed on 3.2% ± 4.5%, 0%, and 1.8% ± 1.2% CD34+ blood cells, respectively. However, after 24 hours in liquid culture with medium alone, or with tumor-necrosis-factor- (TNF-), or with allogeneic mononuclear cells 10.8% ± 3.8%, 75.3% ± 15.0% and 53.7% ± 17.0% CD34+ blood cells, respectively, became CD40+. After incubation for 24 hours with TNF- CD34+CD40+ blood cells expressed only myeloid markers and contained less than 5% CD86+ and CD80+ cells. Also, a 24-hour priming with TNF- or ligation of CD40 significantly increased the CD34+ blood cells alloantigen presenting function. Finally, purified CD34+CD40+ blood cells stimulated an alloreactive T-cell response in MLC, were enriched in granulocytic, monocytic, and dendritic precursors, and generated high numbers of DC in 11-14 d liquid cultures with GM-CSF, SCF, TNF- and FLT-3L. In contrast, CD34+CD40− cells were poorly immunogenic, contained committed granulocytic and erythroid precursors and early progenitors, and differentiated poorly toward the DC lineage. In conclusion, a short incubation with TNF- allows the selection of CD40+ blood progenitors, which may be a useful source of DC precursors for antitumor vaccine studies, and also a CD34+CD40− blood cell fraction that could be exploited in innovative strategies of allogeneic transplantation across HLA barriers.
TECK, an Efficacious Chemoattractant for Human Thymocytes, Uses GPR-9-6/CCR9 as a Specific Receptor
Authors: Youn Byung-S. , Kim Chang H. , Smith Franklin O. , Broxmeyer Hal E.
Published: October 2019

Chemokines regulate leukocytes trafficking in normal and inflammation conditions. Thymus-seeding progenitors are made in bone marrow and migrate to the thymus where they undergo their maturation to antigen-specific T cells. Immature T cells are in thymic cortex, while mature thymocytes are in medulla. Chemokines may be important for homing of thymus-seeding progenitors, and/or differential thymocyte localization in thymus. Here we report that GPR-9-6, now called CC chemokine receptor 9 (CCR9), is a receptor for thymus-expressed chemokine, TECK. Among a panel of chemokines tested, TECK specifically induced calcium flux in CCR9-expressing cell lines. We also showed that TECK efficaciously induced chemotaxis of immature CD4+CD8+ double-positive, and mature CD4+ and CD8+ single-positive human thymocytes. Our data suggest that TECK/CCR9 interaction may play a pivotal role in T-cell migration in the thymus.
REDK, a novel human regulatory erythroid kinase
Authors: Lord Kenneth A. , Creasy Caretha L. , King Andrew G. , King Caroline , Burns Brian M. , Lee John C. , Dillon Susan B.
Published: October 2019

We have identified a novel regulatory erythroid kinase (REDK) that is homologous to a family of dual-specificity kinases. The yeast homolog of REDK negatively regulates cell division, suggesting a similar function for REDK, which is primarily localized in the nucleus. REDK is present in hematopoietic tissues, such as bone marrow and fetal liver, but the RNA is expressed at significant levels only in erythroid or erythropoietin (EPO)-responsive cells. Two novel forms of cDNA (long and short) for REDK have been isolated that appear to be alternative splice products and imply the presence of polypeptides with differing amino termini. The ratio of short-to-long forms of REDK increases dramatically in CD34+ cells cultured with EPO, suggesting differing regulation and function for each form. REDK is predominantly found in nuclear, rather than cytoplasmic, protein extracts, and immunoprecipitated REDK is active in phosphorylating histones H2b, H3, myelin basic protein, and other coimmunoprecipitated proteins. Antisense REDK oligonucleotides promote erythroid colony formation by human bone marrow cells, without affecting colony-forming unit (CFU)-GM, CFU-G, or CFU-GEMM numbers. Maximal numbers of CFU-E and burst-forming unit–erythroid were increased, and CFU-E displayed increased sensitivity to suboptimal EPO concentrations. The data indicate that REDK acts as a brake to retard erythropoiesis.
Thrombin activatable fibrinolysis inhibitor and the risk for deep vein thrombosis
Authors: van Tilburg Nico H. , Rosendaal Frits R. , Bertina Rogier M.
Published: October 2019

Thrombin activatable fibrinolysis inhibitor (TAFI, or procarboxypeptidase B) is the precursor of a recently described carboxypeptidase that potently attenuates fibrinolysis. Therefore, we hypothesized that elevated plasma TAFI levels induce a hypofibrinolytic state associated with an increased risk for venous thrombosis. To evaluate this hypothesis, we developed an electroimmunoassay for TAFI antigen and used this assay to measure TAFI levels in the Leiden Thrombophilia Study, a case–control study of venous thrombosis in 474 patients with a first deep vein thrombosis and 474 age- and sex-matched control subjects. In 474 healthy control subjects, an increase of TAFI with age was observed in women but not in men. Oral contraceptive use also increased the TAFI concentration. TAFI levels above the 90th percentile of the controls (> 122 U/dL) increased the risk for thrombosis nearly 2-fold compared with TAFI levels below the 90th percentile (odds ratio, 1.7; 95% confidence interval, 1.1-2.5). Adjustment for various possible confounders did not materially affect this estimate. These results indicate that elevated TAFI levels form a mild risk factor for venous thrombosis. Such levels were found in 9% of healthy controls and in 14% of patients with a first deep vein thrombosis. Elevated TAFI levels did not enhance the thrombotic risk associated with factor V Leiden but may interact with high factor VIII levels.
Role of IL-16 in delayed-type hypersensitivity reaction
Authors: Yoshimoto Takayuki , Wang Chrong-Reen , Yoneto Toshihiko , Matsuzawa Akio , Cruikshank William W. , Nariuchi Hideo
Published: October 2019

Interleukin (IL)-16 is a chemoattractant cytokine for CD4+ leukocytes. Because delayed-type hypersensitivity (DTH) reaction is mediated by T helper 1 (Th1) cells and CD4+ T cells can be chemoattracted by IL-16, we have investigated the involvement of IL-16 in the DTH reaction. Immunohistochemical analysis revealed the IL-16 expression in infiltrating cells and epithelial cells in the DTH footpads. The IL-16 expression was also detected intracellularly in the infiltrating cells. In addition, markedly increased production of IL-16 was detected in the DTH footpad extracts, but not in the control footpad extracts, by an enzyme-linked immunosorbent assay and also by Western blot analysis. The DTH footpad extracts exhibited a strong chemoattractant activity toward splenic T cells, which was significantly inhibited by the inclusion of neutralizing monoclonal antibody (mAb) against IL-16 in the migration assay. Furthermore, treatment of sensitized mice in vivo with the anti-IL-16 neutralizing mAb significantly suppressed the footpad swelling induced by an antigen challenge, together with decreased infiltration of leukocytes including not only CD4+ T cells but also CD8+ T cells and macrophages into the DTH footpads. Decreased production of macrophage inflammatory protein 1 was also observed in the DTH footpad extracts by the mAb treatment. These results suggest that IL-16 plays an important role in the recruitment of leukocytes—presumably including antigen-specific Th1 cells, which secrete cytokines and chemokines mediating the following hypersensitivity reaction after activation by the interaction with Langerhans cells carrying the antigen—for the elicitation of DTH response.
Increased lipoprotein (a) levels as an independent risk factor for venous thromboembolism
Authors: von Depka Mario , Nowak-Göttl Ulrike , Eisert Roswith , Dieterich Christian , Barthels Monika , Scharrer Inge , Ganser Arnold , Ehrenforth Silke
Published: October 2019

Elevation of serum lipoprotein (a) (Lp[a]) is a known risk factor predisposing to cardiovascular and cerebrovascular disease. However, little is known about the role of increased Lp(a) in venous thromboembolism (VTE). This study evaluated the role of Lp(a) among a panel of established hereditary thrombogenic defects in patients with VTE. A total of 685 consecutive patients with at least one episode of VTE and 266 sex- and age-matched healthy controls were screened with regard to activated protein C resistance, protein C, protein S, and antithrombin deficiency, elevated serum levels of Lp(a), and the factor V G1691A, MTHFR C677T, and prothrombin G20210A mutations. Elevated Lp(a) levels above 30 mg/dL were found in 20% of all patients, as compared to 7% among healthy controls (P < .001, odds ratio [OR] 3.2, 95% confidence interval [CI], 1.9-5.3). The coexistence of FV G1691A and elevated Lp(a) was significantly more prevalent among patients with VTE than in the control group (7% versus 0.8%; P < .001, OR 9.8, 95% CI, 2.4-40.7). No other established prothrombotic risk factor was found to be significantly combined with increased Lp(a). These data suggest that Lp(a) concentrations greater than 30 mg/dL are a frequent and independent risk factor for VTE. Furthermore, elevated Lp(a) levels might contribute to the penetrance of thromboembolic disease in subjects being affected by other prothrombotic defects, such as FV G1691A mutation.