bluuebunny/crossref_metadata_2025 huggingface dataset:
Hemochromatosis genes and other factors contributing to the pathogenesis of porphyria cutanea tarda
Authors: Bulaj Zaneta J. , Phillips John D. , Ajioka Richard S. , Franklin Michael R. , Griffen Linda M. , Guinee Donald J. , Edwards Corwin Q. , Kushner James P.
Published: October 2019

Inherited and acquired factors have been implicated in the pathogenesis of porphyria cutanea tarda (PCT), a disorder characterized by a photosensitive dermatosis and hepatic siderosis. This study, comprising 108 patients with PCT, was intended to define the role of hemochromatosis gene (HFE) mutations in the expression of PCT and to determine the contribution of acquired factors including alcohol, hepatitis C virus (HCV), and estrogen. The 2 known HFEmutations, cysteine 282 tyrosine (Cys282Tyr) and histidine 63 asparagine (His63Asp), were detected by polymerase chain reaction, and anti-HCV immunoglobulin G was detected serologically. Liver biopsies were graded for iron content, inflammation, and fibrosis. Estimates of alcohol and estrogen use were based on a questionnaire. Of the PCT patients tested, 19% were homozygous for the Cys282Tyr mutation; controls were equal to 0.5%. The compound heterozygous genotype was detected in 7% of the PCT patients; controls were less than 1%. The transferrin saturation, serum ferritin, and liver iron burden of all PCT patients were higher than those of nonporphyric controls. The highest values were found in PCT patients homozygous for the Cys282Tyr mutation. Of the patients studied, 59% were HCV positive (compared with 1.8% of the population), and 46% consumed more than 70 g of alcohol daily. Of the female patients, 63% were ingesting estrogens. Hepatic damage was most marked in patients with the Cys282Tyr/Cys282Tyr genotype who had HCV and drank heavily. Homozygosity for the Cys282Tyrmutation and HCV are the greatest risk factors for expression of PCT, and in most patients, more than 1 risk factor was identified. It was common for patients with HCV to consume alcohol. Patients with PCT should be screened for HFE mutations and for HCV.
The anticoagulant factor, protein S, is produced by cultured human vascular smooth muscle cells and its expression is up-regulated by thrombin
Authors: Benzakour Omar , Kanthou Chryso
Published: October 2019

The anticoagulant factor protein S is a secreted vitamin K-dependent γ-carboxylated protein that is mainly made in the liver. Protein S is homologous to the growth arrest specific protein, Gas6, the expression of which is up-regulated in cultured fibroblasts upon serum withdrawal. We report here the synthesis and secretion of protein S by cultured human vascular smooth muscle cells (HVSMCs). Western blot analysis revealed that similar amounts of protein S are secreted by both growing and growth-arrested HVSMCs. HVSMC-derived protein S was found to be γ-carboxylated as it was precipitated by barium citrate and was shown to possess protein C cofactor activity. Treatment with the vitamin K antagonist warfarin led to the accumulation of intracellular undercarboxylated protein S forms that were rapidly secreted upon the reintroduction of vitamin K. Northern blotting analysis showed that cultured HVSMCs express a protein S transcript. The expression of protein S messenger RNA was unaffected by either warfarin, growth arrest, or various VSMC mitogens, such as platelet-derived growth factor-BB, basic fibroblast growth factor, transforming growth factor-β, or hepatocyte growth factor. Thrombin, however, induced an up-regulation of protein S expression at both messenger RNA and protein levels. The evidence we provide for protein S secretion by cultured HVSMCs and its up-regulation by thrombin, together with earlier reports showing that protein S acts as a mitogen for these cells, suggests that, in addition to its known role in regulating blood clotting, protein S may also be an important autocrine factor in the pathophysiology of the vasculature.
Allogeneic hematopoietic chimerism in mice treated with sublethal myeloablation and anti-CD154 antibody: absence of graft-versus-host disease, induction of skin allograft tolerance, and prevention of recurrent autoimmunity in islet-allografted NOD/Lt mice
Authors: Seung Edward , Iwakoshi Neal , Woda Bruce A. , Markees Thomas G. , Mordes John P. , Rossini Aldo A. , Greiner Dale L.
Published: October 2019

We describe a tolerance-based stem cell transplantation protocol that combines sublethal radiation with transient blockade of the CD40-CD154 costimulatory pathway using an anti-CD154 antibody. With this protocol, we established hematopoietic chimerism in BALB/c mice transplanted with fully allogeneic C57BL/6 bone marrow. The percentage of donor-origin mononuclear cells in recipients was more than 99%. In addition, all chimeric mice treated with anti-CD154 antibody remained free of graft-versus-host disease (GVHD) and accepted donor-origin but not third-party skin allografts. It was similarly possible to create allogeneic hematopoietic chimerism in NOD/Lt mice with spontaneous autoimmune diabetes. Pancreatic islet allografts transplanted into chimeric NOD/Lt mice were resistant not only to allorejection but also to recurrence of autoimmunity. We conclude that it is possible to establish robust allogeneic hematopoietic chimerism in sublethally irradiated mice without subsequent GVHD by blocking the CD40-CD154 costimulatory pathway using as few as 2 injections of anti-CD154 antibody. We also conclude that chimerism created in this way generates donor-specific allograft tolerance and reverses the predisposition to recurrent autoimmune diabetes in NOD/Lt mice, enabling them to accept curative islet allografts.
Pharmacologic doses of granulocyte colony-stimulating factor affect cytokine production by lymphocytes in vitro and in vivo
Authors: Sloand Elaine M. , Kim Sonnie , Maciejewski Jaroslaw P. , Van Rhee Fritz , Chaudhuri Aniruddho , Barrett John , Young Neal S.
Published: October 2019

Peripheral blood stem cell (PBSC) transplantation is successful in improving engraftment without increasing acute graft-versus-host disease (GVHD), despite much larger numbers of T cells in unmanipulated PBSCs than in bone marrow grafts. In mouse models and retrospective human studies, granulocyte colony-stimulating factor (G-CSF) therapy has been associated with less acute GVHD. We studied the effect of G-CSF on interferon (IFN)-γ and IL-4 expression in CD4+lymphocytes. CD4+ cells co-cultivated with G-CSF and stimulated with PHA or CD3 monoclonal antibodies showed significant decreases in IFN-γ and increases in IL-4 expression (n = 13;P < .01). G-CSF appeared to have a direct effect on CD4+ cells independent of monocytes present in the culture because purified CD4+ cells exposed to G-CSF, washed, and cocultivated with untreated monocytes demonstrated similar changes in IFN-γ and IL-4 expression, whereas untreated CD4+ cells cocultured with G-CSF–stimulated monocytes behaved as controls. We then studied peripheral blood mononuclear cells (PBMCs) from G-CSF–mobilized PBSC donors. When their PBMCs were cultured with PHA or CD3 monoclonal antibody, the percent of IFN-γ–expressing cells decreased by a mean of 55% and 42%, respectively, whereas the percent of IL-4–containing cells increased by a mean of 39% and 58%, respectively, following G-CSF stimulation. Increased apoptosis of IFN-γ–producing CD4+ cells was not responsible for the shift in TH1/TH2 subsets. G-CSF-R mRNA was present in both CD4+ and CD8+ cells. These results suggest that G-CSF decreases IFN-γ and increases IL-4 production in vitro and in vivo and likely modulates a balance between TH1 and TH2 cells, an effect that may be important in PBSC transplantation.
Vascular endothelial growth factor synergistically enhances bone morphogenetic protein-4-dependent lymphohematopoietic cell generation from embryonic stem cells in vitro
Authors: Nakayama Naoki , Lee Jae , Chiu Laura
Published: October 2019

The totipotent mouse embryonic stem (ES) cell is known to differentiate into cells expressing the β-globin gene when stimulated with bone morphogenetic protein (BMP)-4. Here, we demonstrate that BMP-4 is essential for generating both erythro-myeloid colony-forming cells (CFCs) and lymphoid (B and NK) progenitor cells from ES cells and that vascular endothelial growth factor (VEGF) synergizes with BMP-4. The CD45+ myelomonocytic progenitors and Ter119+ erythroid cells began to be detected with 0.5 ng/mL BMP-4, and their levels plateaued at approximately 2 ng/mL. VEGF alone weakly elevated the CD34+ cell population though no lymphohematopoietic progenitors were induced. However, when combined with BMP-4, 2 to 20 ng/mL VEGF synergistically augmented the BMP-4-dependent generation of erythro-myeloid CFCs and lymphoid progenitors from ES cells, which were enriched in CD34+ CD31lo and CD34+CD45− cell populations, respectively, in a dose-dependent manner. Furthermore, during the 7 days of in vitro differentiation, BMP-4 was required within the first 4 days, whereas VEGF was functional after the action of BMP-4 (in the last 3 days). Thus, VEGF is a synergistic enhancer for the BMP-4-dependent differentiation processes, and it seems to be achieved by the ordered action of the 2 factors.
Expansion of hematopoietic stem cells in the developing liver of a mouse embryo
Authors: Ema Hideo , Nakauchi Hiromitsu
Published: October 2019

The activity of hematopoietic stem cells in the developing liver of a C57BL/6 mouse embryo was quantified by a competitive repopulation assay. Different doses of fetal liver cells at days 11 to 18 of gestation were transplanted into irradiated mice together with 2 × 105 adult bone marrow cells. A long-term repopulation in myeloid-, B-cell, and T-cell lineage by fetal liver cells was evaluated at 20 weeks after transplantation. At day 12 of gestation multilineage repopulating activity was first detected in the liver as 50 repopulating units (RU) per liver. The number of RU per liver increased 10-fold and 33-fold by day 14 and day 16 of gestation, and decreased thereafter, suggesting a single wave of stem cell development in the fetal liver. A limiting dilution analysis revealed that the frequency of competitive repopulating units (CRU) in fetal liver cells at day 12 of gestation was similar to that at day 16 of gestation. Because of an increase of total fetal liver cell number, the absolute number of CRU per liver from days 12 to 16 of gestation increased 38-fold. Hence, the mean activity of stem cells (MAS) that is given by RU per CRU remained constant from days 12 to 16 of gestation. From these data we conclude that hematopoietic stem cells expand in the fetal liver maintaining their level of repopulating potential.
Delineation of a minimal interval and identification of 9 candidates for a tumor suppressor gene in malignant myeloid disorders on 5q31
Authors: Horrigan Stephen K. , Arbieva Zarema H. , Xie Hong Yan , Kravarusic Jelena , Fulton Noreen C. , Naik Haley , Le Tiffany T. , Westbrook Carol A.
Published: October 2019

Interstitial deletion or loss of chromosome 5 is frequent in malignant myeloid disorders, including myelodysplasia (MDS) and acute myeloid leukemia (AML), suggesting the presence of a tumor suppressor gene. Loss of heterozygosity (LOH) analysis was used to define a minimal deletion interval for this gene. Polymorphic markers on 5q31 were identified using a high-resolution physical and radiation hybrid breakpoint map and applied to a patient with AML with a subcytogenetic deletion of 5q. By comparing the DNA from leukemic cells to buccal mucosa cells, LOH was detected with markers D5S476 and D5S1372 with retention of flanking markers D5S500 to D5S594. The D5S500–D5S594 interval, which covers approximately 700 kb, thus represents a minimal localization for the tumor suppressor gene. Further refinement of the physical map enabled the specification of 9 transcription units within the encompassing radiation hybrid bins and 7 in flanking bins. The 9 candidates include genes CDC25, HSPA9, EGR1, CTNNA1, and 5 unknown ESTs. Reverse-transcription polymerase chain reaction confirms that all of them are expressed in normal human bone marrow CD34+ cells and in AML cell lines and thus represent likely candidates for the MDS–AML tumor suppressor gene at 5q31.
Lysophosphatidic acid opens a Ca++ channel in human erythrocytes
Authors: Yang Lu , Andrews Dina A. , Low Philip S.
Published: October 2019

Lysophosphatidic acid (LPA) is a lipid-derived second messenger that mobilizes many cells of the circulatory and vascular systems to assist in thrombus development and wound healing. LPA, however, has not been tested on human erythrocytes, largely because erythrocytes are considered to be both biologically inert and inactive in intercellular communication. To test this presumption, we have examined the impact of LPA on signaling reactions within the human red blood cell (RBC). Using both 45Ca++ and a Ca++-sensitive fluorescent probe (Fluo-3), we demonstrated that LPA, but not phosphatidic acid or the closely related sphingosine-1–phosphate, stimulates the influx of micromolar quantities of extracellular Ca++ into fresh RBCs. This Ca++ influx was shown to be channel mediated rather than leak promoted because the influx was observed at LPA concentrations too low to perturb membrane integrity, it was inhibited by P-type but not L-type Ca++ channel blockers, it was inhibited by broad-specificity protein kinase inhibitors, and it was not induced by inactive analogues of LPA. Further characterization reveals that only approximately 25% of the RBCs participate in LPA-induced Ca++ entry and that within this active population, Ca++ gating occurs in an all-or-nothing manner. Because the stimulation of Ca++ uptake occurs at LPA concentrations (1-5 μmol/L) known to occur near a developing thrombus and because the internalized Ca++can potentially promote prothrombic properties in the stimulated RBCs, we conclude that RBCs are not insensitive to signals released from other cells.
Acute onset hemoglobinemia and/or hemoglobinuria and sequelae following Rho(D) immune globulin intravenous administration in immune thrombocytopenic purpura patients
Authors: Gaines Ann Reed
Published: October 2019

Rho(D) immune globulin intravenous (anti-D IGIV) was licensed by the United States Food and Drug Administration (FDA) in March 1995 to treat patients with immune thrombocytopenic purpura (ITP). Anti-D IGIV induces extravascular hemolysis, an expected adverse reaction that is consistent with the presumed mechanism of action. Between licensure and April 1999, the FDA received 15 reports of hemoglobinemia and/or hemoglobinuria following anti-D IGIV administration that met the case definition for this review. The mechanism responsible for hemoglobinemia and/or hemoglobinuria is unexplained. Review of these reports was prompted by the seriousness and the unexpectedness of treatment-associated sequelae experienced by 11 patients. Of these patients, 7 developed sufficient onset or exacerbation of anemia that orders were written for packed red blood cell transfusions, although only 6 patients were transfused. Eight patients experienced the onset or exacerbation of renal insufficiency, and 2 patients underwent dialysis. One patient died due to complications of exacerbated anemia. Six patients experienced 2 to 3 sequelae. Absent validated incidence data, a 1.5% estimated incidence rate from published clinical trial data and a 0.1% estimated reporting rate from FDA and drug utilization data were calculated for reported cases of hemoglobinemia and/or hemoglobinuria. This review presents the first case series of anti-D-IGIV–associated hemoglobinemia and/or hemoglobinuria and provides pretreatment and posttreatment clinical and laboratory findings of the case series patients. The primary purpose of this review is to increase awareness of this potentially serious occurrence among physicians and health care professionals who manage ITP patients treated with anti-D IGIV, thereby enabling prompt recognition and treatment of sequelae.
Improved muscle-derived expression of human coagulation factor IX from a skeletal actin/CMV hybrid enhancer/promoter
Authors: Hagstrom J. Nathan , Couto Linda B. , Scallan Ciaran , Burton Melissa , McCleland Mark L. , Fields Paul A. , Arruda Valder R. , Herzog Roland W. , High Katherine A.
Published: October 2019

Hemophilia B is caused by the absence of functional coagulation factor IX (F.IX) and represents an important model for treatment of genetic diseases by gene therapy. Recent studies have shown that intramuscular injection of an adeno-associated viral (AAV) vector into mice and hemophilia B dogs results in vector dose–dependent, long-term expression of biologically active F.IX at therapeutic levels. In this study, we demonstrate that levels of expression of approximately 300 ng/mL (6% of normal human F.IX levels) can be reached by intramuscular injection of mice using a 2- to 4-fold lower vector dose (1 × 1011 vector genomes/mouse, injected into 4 intramuscular sites) than previously described. This was accomplished through the use of an improved expression cassette that uses the cytomegalovirus (CMV) immediate early enhancer/promoter in combination with a 1.2-kilobase portion of human skeletal actin promoter. These results correlated with enhanced levels of F.IX transcript and secreted F.IX protein in transduced murine C2C12 myotubes. Systemic F.IX expression from constructs containing the CMV enhancer/promoter alone was 120 to 200 ng/mL in mice injected with 1 × 1011vector genomes. Muscle-specific promoters performed poorly for F.IX transgene expression in vitro and in vivo. However, the incorporation of a sequence from the -skeletal actin promoter containing at least 1 muscle-specific enhancer and 1 enhancer-like element further improved muscle-derived expression of F.IX from a CMV enhancer/promoter-driven expression cassette over previously published results. These findings will allow the design of a clinical protocol for therapeutic levels of F.IX expression with lower vector doses, thus enhancing efficacy and safety of the protocol.
Induction of apoptosis by extracellular ubiquitin in human hematopoietic cells: possible involvement of STAT3 degradation by proteasome pathway in interleukin 6-dependent hematopoietic cells
Authors: Daino Hanako , Matsumura Itaru , Takada Koji , Odajima Junko , Tanaka Hirokazu , Ueda Shuji , Shibayama Hirohiko , Ikeda Hirokazu , Hibi Masahiko , Machii Takashi , Hirano Toshio , Kanakura Yuzuru
Published: October 2019

The ubiquitin–proteasome pathway is responsible for selective degradation of short-lived cellular proteins and is critical for the regulation of many cellular processes. We previously showed that ubiquitin (Ub) secreted from hairy cell leukemia cells had inhibitory effects on clonogenic growth of normal hematopoietic progenitor cells. In this study, we examined the effects of exogenous Ub on the growth and survival of a series of human hematopoietic cells, including myeloid cell lines (HL-60 and U937), a B-cell line (Daudi), and T-cell lines (KT-3, MT-4, YTC-3, and MOLT-4). Exogenous Ub inhibited the growth of various hematopoietic cell lines tested, especially of KT-3 and HL-60 cells. The growth-suppressive effects of Ub on KT-3 and HL-60 cells were almost completely abrogated by the proteasome inhibitor PSI or MG132, suggesting the involvement of the proteasome pathway in this process. Furthermore, exogenous Ub evoked severe apoptosis of KT-3 and HL-60 cells through the activation of caspase-3. In interleukin-6 (IL-6)-dependent KT-3 cells, STAT3 was found to be conjugated by exogenous biotinylated Ub and to be degraded in a proteasome-dependent manner, whereas expression levels of STAT1, STAT5, or mitogen-activated protein kinase were not affected. Moreover, IL-6-induced the up-regulation of Bcl-2 and c-myc, and JunB was impaired in Ub-treated KT-3 cells, suggesting that the anti-apoptotic and mitogenic effects of IL-6 were disrupted by Ub. These results suggest that extracellular Ub was incorporated into hematopoietic cells and mediated their growth suppression and apoptosis through proteasome-dependent degradation of selective cellular proteins such as STAT3.
Transcriptional control of the human plasma membranephospholipid scramblase 1 gene is mediated by interferon-α
Authors: Zhou Quansheng , Zhao Ji , Al-Zoghaibi Fahad , Zhou Aimin , Wiedmer Therese , Silverman Robert H. , Sims Peter J.
Published: October 2019

Interferons (IFNs) mediate their diverse biologic activities through induction of the expression of multiple genes. Whereas the mode of action of certain of these IFN-regulated genes has been well characterized, most of the molecular and cellular events underlying the constellation of biologic responses to the IFNs remain unresolved. This study showed that the newly identified PLSCR1 gene for phospholipid scramblase, previously implicated in remodeling of plasma membrane phospholipids, is regulated at the transcriptional level by IFN-. Analysis of 5′ flanking genomic sequence in reporter constructs showed that transcriptional control of PLSCR1 was entirely regulated by a single IFN-stimulated response element located in the first exon. A similar induction of PLSCR1 by IFN-2a was also observed in a variety of other human tumor cell lines as well as in human umbilical vein endothelial cells. In these cell lines, the marked IFN-2a–induced increase in PLSCR1 protein expression, ranging as high as 10-fold above basal levels, was not accompanied by increased cell surface exposure of phosphatidylserine, suggesting that remodeling of the cell surface requires both exposure to IFN and a second yet-to-be identified event to stimulate plasma membrane phospholipid scramblase activity and to mobilize phosphatidylserine to the cell surface.
Isolation and characterization of an acute promyelocytic leukemia cell line selectively resistant to the novel antileukemic and apoptogenic retinoid 6-[3-adamantyl-4-hydroxyphenyl]-2-naphthalene carboxylic acid
Authors: Ponzanelli Isabella , Giannı̀ Maurizio , Giavazzi Raffaella , Garofalo Angela , Nicoletti Ines , Reichert Uwe , Erba Eugenio , Rambaldi Alessandro , Terao Mineko , Garattini Enrico
Published: October 2019

6-[3-adamantyl-4-hydroxyphenyl]-2-naphthalene carboxylic acid (CD437) is a novel compound that represents the prototype of a new class of synthetic retinoids with apoptogenic properties in acute promyelocytic leukemia (APL) and other types of leukemia. In this article, using SCID mice xenografted with APL-derived NB4 cells, we demonstrate that CD437 has significant antileukemic activity in vivo. In addition, we report on the isolation and characterization of an APL cell line (NB4.437r) resistant to CD437. The cell line retains expression of PML-RAR and is approximately 33-fold more resistant than the parental counterpart to the apoptogenic effects of the retinoid. Resistance is relatively specific to CD437 and structural congeners because the NB4.437r cell line is still sensitive to various types of apoptogenic compounds. The CD437-resistant cell line maintains sensitivity to the antiproliferative and apoptotic action of all-trans-retinoic acid, AM580, and fenretinide, though it shows partial resistance to the cytodifferentiating effects of the first 2 compounds. Resistance to CD437 lays upstream of the CD437-induced release of cytochrome c from the mitochondria and the activation of caspase-3, -7, -8, and -9. Furthermore, NB4.437r cells are deficient in the CD437-dependent activation of nuclear NFkb and AP1-binding activities and in the phosphorylation of the protein kinase Akt. In the case of AP1, deficient assembly of the complex is not caused by the lack of activation of the Jun N-terminal kinase (JNK) family of kinases. The novel cell line will be useful in the elucidation of the molecular mechanisms underlying the apoptogenic action of CD437 and structurally related retinoids.
Survival of donor cells 25 years after intrauterine transfusion
Authors: Viëtor Henk E. , Hallensleben Eric , van Bree Simone P. M. J. , van der Meer Ellen M. W. , Kaal Suzanne E. J. , Bennebroek-Gravenhorst Jack , Kanhai Humphrey H. H. , Brand Anneke , Claas Frans H. J.
Published: October 2019

Persistence of donor leukocytes in the circulation of recipients of intrauterine transfusion (IUT) has been observed up to 5 years after birth. The aim of this study was to determine whether transfusions with nonirradiated, nonleukocyte-depleted donor blood during the fetal period resulted in long-term immunomodulation of the recipient. Twenty-four surviving IUT recipients between 1966 and 1976 were tested for autoimmune disease and autoantibodies at follow-up. Ten had sex-mismatched donors and were therefore informative for chimerism studies using fluorescence in situ hybridization (FISH). Seven female recipients could be tested for chimerism using a Y- chromosome–specific polymerase chain reaction (PCR) because they received at least 1 IUT from a male donor. Nine recipients could be studied for cytotoxic T-lymphocyte precursor (CTLp) and helper T-lymphocyte precursor (HTLp) frequencies because the original donors were available for testing. All surviving IUT recipients were in good health at the time of the examination, and routine laboratory testing revealed no abnormalities. None of the IUT recipients were chimeric as determined by FISH analysis, but Y-chromosome–specific sequences were detected by PCR in 6 of the 7 women. However, the CTLp and HTLp frequencies of the IUT recipients against the donors were comparable to those of the controls. The current study provides evidence that IUT can result in the persistence of donor cells in the recipient for a period longer than 20 years but that it is not associated with immunotolerance or with signs of chronic antigenic stimulation.
Novel nuclear target for thrombin: activation of the Elk1 transcription factor leads to chemokine gene expression
Authors: Li Qi-Jing , Vaingankar Sucheta , Sladek Frances M. , Martins-Green Manuela
Published: October 2019

Thrombin is primarily known for its role in homeostasis and thrombosis. However, this enzyme also plays important roles in wound healing and pathologic situations such as inflammation and tumorigenesis. Among the molecules stimulated by thrombin in these latter processes are the stress response proteins, chemokines. Chemokines are also known for their roles in inflammatory responses and tumor development. These correlative observations strongly suggest that chemokines may be mediators of some of thrombin's functions in these processes. Elucidation of the molecular mechanisms of stimulation of chemokines by thrombin may help to unravel the ways in which their expression can be modulated. Up-regulation of the chemokine 9E3/cCAF by thrombin occurs via its proteolytically activated receptor with subsequent transactivation of the epidermal growth factor receptor tyrosine kinase. This study shows that stimulation by thrombin very rapidly activates this chemokine at the transcriptional level, that 2 Elk1 binding elements located between −534 and −483 bp of the promoter are major thrombin response elements, that activation occurs via the Elk1 transcription factor, and that the latter is directly activated by MEK1/ERK2. The common occurrence of Elk1 binding domains in the promoters of immediate early response genes suggests that it may be characteristically involved in gene activation by stress-inducing agents.
Endogenous biosynthesis of thromboxane and prostacyclin in 2 distinct murine models of atherosclerosis
Authors: Praticò Domenico , Cyrus Tillmann , Li Hongwei , FitzGerald Garret A.
Published: October 2019

Thromboxane A2 is a potent vasoconstrictor and platelet agonist; prostacyclin is a potent platelet inhibitor and vasodilator. Altered biosynthesis of these eicosanoids is a feature of human hypercholesterolemia and atherosclerosis. This study examined whether in 2 murine models of atherosclerosis their levels are increased and correlated with the evolution of the disease. Urinary 2,3-dinor thromboxane B2 and 2,3-dinor-6-keto prostaglandin F1α, metabolites of thromboxane and prostacyclin, respectively, were assayed in apoliprotein E (apoE)-deficient mice on chow and low-density lipoprotein receptor (LDLR)-deficient mice on chow and a Western-type diet. Atherosclerosis lesion area was measured by en face method. Both eicosanoids increased in apoE-deficient mice on chow and in LDLR-deficient mice on a high-fat diet, but not in LDLR-deficient mice on chow by the end of the study. Aspirin suppressed ex vivo platelet aggregation, serum thromboxane B2, and 2,3-dinor thromboxane B2, and significantly reduced the excretion of 2,3-dinor-6-keto prostaglandin F1α in these animals. This study demonstrates that thromboxane as well as prostacyclin biosynthesis is increased in 2 murine models of atherogenesis and is secondary to increased in vivo platelet activation. Assessment of their generation in these models may afford the basis for future studies on the functional role of these eicosanoids in the evolution and progression of atherosclerosis.
Successful stem cell transplantation following orthotopic liver transplantation from the same haploidentical family donor in a girl with hemophagocytic lymphohistiocytosis
Authors: Matthes-Martin Susanne , Peters Christina , Königsrainer Alfred , Fritsch Gerhard , Lion Thomas , Heitger Andreas , Kapelari Klaus , Kronberger Martina , Offner Felix , Wrba Fritz , Margreiter Raimund , Gadner Helmut
Published: October 2019

The case of a 4-month-old girl with familial hemophagocytic lymphohistiocytosis is described. The patient underwent stem cell transplantation from her haploidentical mother 2 months after receiving a living-related liver transplant from the same donor for acute hepatic failure. Conditioning regimen consisted of 16 mg/kg busulfan, 200 mg/kg cyclophosphamide, 10 mg/kg thiothepa, and antithymocyte globulin. Myeloid engraftment occurred on day +10, but CD3+ cells of recipient origin remained. To convert the T-cell chimerism, the patient received donor lymphocyte infusion on day +43, and subsequently the allelic pattern changed to complete donor genotype on day +57. Four months after stem cell transplantation the patient is disease free, with complete donor chimerism in bone marrow and stable hepatic graft function without any immunosuppressive therapy.
Functional expression of CCR1, CCR3, CCR4, and CXCR4 chemokine receptors on human platelets
Authors: Clemetson Kenneth J. , Clemetson Jeannine M. , Proudfoot Amanda E. I. , Power Christine A. , Baggiolini Marco , Wells Timothy N. C.
Published: October 2019

Platelets are known to contain platelet factor 4 and β-thromboglobulin, α-chemokines containing the CXC motif, but recent studies extended the range to the β-family characterized by the CC motif, including RANTES and Gro-α. There is also evidence for expression of chemokine receptors CCR4 and CXCR4 in platelets. This study shows that platelets have functional CCR1, CCR3, CCR4, and CXCR4 chemokine receptors. Polymerase chain reaction detected chemokine receptor messenger RNA in platelet RNA. CCR1, CCR3, and especially CCR4 gave strong signals; CXCR1 and CXCR4 were weakly positive. Flow cytometry with specific antibodies showed the presence of a clear signal for CXCR4 and weak signals for CCR1 and CCR3, whereas CXCR1, CXCR2, CXCR3, and CCR5 were all negative. Immunoprecipitation and Western blotting with polyclonal antibodies to cytoplasmic peptides clearly showed the presence of CCR1 and CCR4 in platelets in amounts comparable to monocytes and CCR4 transfected cells, respectively. Chemokines specific for these receptors, including monocyte chemotactic protein 1, macrophage inflammatory peptide 1α, eotaxin, RANTES, TARC, macrophage-derived chemokine, and stromal cell–derived factor 1, activate platelets to give Ca++ signals, aggregation, and release of granule contents. Platelet aggregation was dependent on release of adenosine diphosphate (ADP) and its interaction with platelet ADP receptors. Part, but not all, of the Ca++ signal was due to ADP release feeding back to its receptors. Platelet activation also involved heparan or chondroitin sulfate associated with the platelet surface and was inhibited by cleavage of these glycosaminoglycans or by heparin or low molecular weight heparin. These platelet receptors may be involved in inflammatory or allergic responses or in platelet activation in human immunodeficiency virus infection.
Evolving characteristics of AIDS-related lymphoma
Authors: Levine Alexandra M. , Seneviratne Lasika , Espina Byron M. , Wohl Amy Rock , Tulpule Anil , Nathwani Bharat N. , Gill Parkash S.
Published: October 2019

Over time, the epidemiologic and demographic characteristics of AIDS have changed in the United States, while the use of highly active antiretroviral therapy has changed the natural history of the disease. The goal of the study was to ascertain any changes in the epidemiologic, immunologic, pathologic, or clinical characteristics of AIDS-related lymphoma (ARL) over the course of the AIDS epidemic. Records of 369 patients with ARL diagnosed or treated at a single institution from 1982 through 1998 were reviewed. Single institutional data were compared to population-based data from the County of Los Angeles. Significant changes in the demographic profile of patients with newly diagnosed ARL have occurred, with the later time intervals associated with a higher prevalence in women (P = .25), in Latino/Hispanic individuals (P < .0001), and in those who acquired human immunodeficiency virus (HIV) heterosexually (P = .01). These changes were similar in both countywide, population-based analyses and in those from the single institution. The median CD4+ lymphocyte count at lymphoma diagnosis has decreased significantly over the years, from 177/dL in the earliest time period (1982-1986), to 53/dL in the last time period from 1995 to 1998 (P = .0006). The pathologic spectrum of disease has also changed, with a decrease in the prevalence of small noncleaved lymphoma (P = .0005) and an increase in diffuse large cell lymphoma (P < .0001). Despite changes in the use of antiretroviral or chemotherapy regimens, the median survival has not significantly changed.
Engraftment and survival after unrelated-donor bone marrow transplantation: a report from the National Marrow Donor Program
Authors: Davies Stella M. , Kollman Craig , Anasetti Claudio , Antin Joseph H. , Gajewski James , Casper James T. , Nademanee Auayporn , Noreen Harriet , King Roberta , Confer Dennis , Kernan Nancy A.
Published: October 2019

We analyzed engraftment of unrelated-donor (URD) bone marrow in 5246 patients who received transplants facilitated by the National Marrow Donor Program between August 1991 and June 1999. Among patients surviving at least 28 days, 4% had primary graft failure (failure to achieve an absolute neutrophil count > 5 × 108/L before death or second stem-cell infusion). Multivariate logistic regression analysis showed that engraftment was associated with marrow matched at HLA-A, HLA-B, and DRB1; higher cell dose; younger recipient; male recipient; and recipient from a non–African American ethnic group. More rapid myeloid engraftment was associated with marrow serologically matched at HLA-A and HLA-B, DRB1 match, higher cell dose (in non-T-cell–depleted cases), younger recipient, recipient seronegativity for cytomegalovirus (CMV), male donor, no methotrexate for graft-versus-host disease prophylaxis, and transplantation done in more recent years. A platelet count higher than 50 × 109/L was achieved by 47% of patients by day 100. Conditional on survival to day 100, survival at 3 years was 61% in those with platelet engraftment at day 30, 58% in those with engraftment between day 30 and day 100, and 33% in those without engraftment at day 100 (P < .0001). Factors favoring platelet engraftment were higher cell dose, DRB1 allele match, recipient seronegativity for CMV, HLA-A and HLA-B serologically matched donor, and male donor. Secondary graft failure occurred in 10% of patients achieving initial engraftment, and 18% of those patients are alive. These data demonstrate that quality of engraftment is an important predictor of survival after URD bone marrow transplantation.
Stable in vivo expression of glucose-6-phosphate dehydrogenase (G6PD) and rescue of G6PD deficiency in stem cells by gene transfer
Authors: Rovira Ana , De Angioletti Maria , Camacho-Vanegas Olga , Liu Delong , Rosti Vittorio , Gallardo Humilidad F. , Notaro Rosario , Sadelain Michel , Luzzatto Lucio
Published: October 2019

Many mutations of the housekeeping gene encoding glucose-6-phosphate dehydrogenase (G6PD) cause G6PD deficiency in humans. Some underlie severe forms of chronic nonspherocytic hemolytic anemia (CNSHA) for which there is no definitive treatment. By using retroviral vectors pseudotyped with the vesicular stomatitis virus G glycoprotein that harbor the human G6PD (hG6PD) complementary DNA, stable and lifelong expression of hG6PD was obtained in all the hematopoietic tissues of 16 primary bone marrow transplant (BMT) recipient mice and 14 secondary BMT recipients. These findings demonstrate the integration of a functional gene in totipotent stem cells. The average total G6PD in peripheral blood cells of these transplanted mice, measured as enzyme activity, was twice that of untransplanted control mice. This allowed the inference that the amount of G6PD produced by the transduced gene must be therapeutically effective. With the same vectors both the cloning efficiency and the ability to form embryoid bodies were restored in embryonic stem cells, in which the G6PD gene had been inactivated by targeted homologous recombination, thus effectively rescuing their defective phenotype. Finally, expression of normal human G6PD in hG6PD-deficient primary hematopoietic cells and in human hematopoietic cells engrafted in nonobese diabetic/severe combined immunodeficient mice was obtained. This approach could cure severe CNSHA caused by G6PD deficiency.
Immunologic dynamics in hemophiliac patients infected with hepatitis C virus and human immunodeficiency virus: influence of antiretroviral therapy
Authors: Yokozaki Shouichi , Takamatsu Junki , Nakano Isao , Katano Yoshiaki , Toyoda Hidenori , Hayashi Kazuhiko , Hayakawa Tetsuo , Fukuda Yoshihide
Published: October 2019

Infection with hepatitis C virus (HCV) or human immunodeficiency virus (HIV) or both is common in hemophiliac patients due to putative transmission through clotting factor concentrates. Recently, highly active antiretroviral therapy (HAART) has been found to markedly improve viremia and immunologic parameters in patients infected with HIV. This report considers interactions between these viral infections, the immune system, and antiretroviral therapy. A total of 130 male hemophiliac patients were grouped according to type of viremia (HCV, HIV, both, or neither). Along with 30 healthy men age-matched to viremic patients, these groups were compared with respect to viral load and immunologic parameters. Thirty-five patients treated as above for HIV were serially followed up. HCV infection was associated with reduced peripheral B-cell and CD4+-cell counts and with increased serum IgG and IgM levels, whereas HIV infection was associated with reduced peripheral CD4+-cell counts and increased serum IgG and IgA levels. In patients with both viruses, HCV and HIV RNA load correlated inversely with peripheral B-cell and CD4+-cell counts, respectively. HAART reduced levels of both viruses in the blood. Of the 25 patients with both viruses, HAART eliminated HCV in 2. In conclusion, immunologic dynamics differed between hemophiliac patients infected with HCV, HIV, or both. The relative dynamics of HCV viral load, peripheral B-cell count, and serum IgM level were similar to those of HIV viral load, CD4+-cell count, and serum IgA.
Interferon α down-regulates telomerase reverse transcriptase and telomerase activity in human malignant and nonmalignant hematopoietic cells
Authors: Xu Dawei , Erickson Sven , Szeps Michael , Gruber Astrid , Sangfelt Olle , Einhorn Stefan , Pisa Pavel , Grandér Dan
Published: October 2019

Recently, the derepressed expression of the catalytic subunit of telomerase, human telomerase reverse transcriptase (hTERT), the enzyme that elongates telomeres, has been implicated as an important step in the immortalization process. The exact regulation of hTERT expression, which is the rate-limiting factor for telomerase activity, is at present unclear. As transformed cells seem to be dependent on a constitutive telomerase activity, the availability of inhibitors would potentially be of great value in antineoplastic therapy. Interferons (IFNs) have been successfully used in the treatment of several forms of malignancies, but the underlying molecular mechanisms responsible for the antitumor activity are poorly defined. In this study we have investigated the effects of IFNs on hTERT expression and telomerase activity. We found that IFN-α rapidly (commonly within 4 hours) and significantly down-regulates the expression of hTERT and telomerase activity in a number of human malignant hematopoietic cell lines, primary leukemic cells from patients with acute leukemia as well as T-lymphocytes from healthy donors. This effect of IFN-α did not seem to depend on IFN-α–mediated cell growth arrest or alterations in c-myc expression. The finding that IFN induces a repression of hTERT and a decrease in telomerase activity suggests a novel mechanism that may play a significant role in the antitumor action of IFN.
Detection of differentially expressed genes in lymphomas using cDNA arrays: identification of clusterin as a new diagnostic marker for anaplastic large-cell lymphomas
Authors: Wellmann Axel , Thieblemont Catherine , Pittaluga Stefania , Sakai Akira , Jaffe Elaine S. , Siebert Paul , Raffeld Mark
Published: October 2019

This study reports the first use of gene array technology for the identification of a tumor-specific marker in lymphoid neoplasms. The differential gene expression of 31 hematopoietic cell lines, representing most major lymphoma subgroups of B- and T-cell origin, was assessed by hybridizing labeled complementary DNA to Atlas human expression arrays containing 588 genes. Genes known to be specific for B, T, or myelomonocytic lineages were appropriately identified in the arrays, validating the general utility of this approach. One gene,clusterin, not previously known to be expressed in lymphoid neoplasms, was specifically found in all 4 anaplastic large-cell lymphoma (ALCL) cell lines, but not in any of the 27 remaining tumor lines. Using a monoclonal antibody against clusterin, its differential expression was confirmed by Western blotting and immunohistochemistry. A total of 198 primary lymphomas (representing most major lymphoma subtypes), including 36 cases of systemic ALCL, were surveyed for clusterin expression by immunohistochemistry and Western blotting. All of the 36 ALCL cases marked for clusterin, with most cases showing moderate to strong staining in the majority of neoplastic cells. Clusterin expression was not related to expression of anaplastic lymphoma kinase-1. With 2 exceptions, none of the remaining 162 non-ALCL cases marked with the clusterin antibody, including Hodgkin disease and primary cutaneous ALCL. In reactive lymphoid tissues, only follicular dendritic cells and fibroblastic reticular cells exhibited staining. Clusterin is a highly conserved glycoprotein implicated in intercellular and cell matrix interactions, regulation of the complement system, lipid transport, stress responses, and apoptosis. Although its function in ALCL is unknown, the unique expression of clusterin within this category of lymphoma provides an additional marker for the diagnosis of ALCL. This study illustrates the enormous potential of gene array technologies for diagnostic marker discovery.
Cubilin P1297L mutation associated with hereditary megaloblastic anemia 1 causes impaired recognition of intrinsic factor–vitamin B12 by cubilin
Authors: Kristiansen Mette , Aminoff Maria , Jacobsen Christian , de la Chapelle Albert , Krahe Ralf , Verroust Pierre J. , Moestrup Søren K.
Published: October 2019

Megaloblastic anemia 1 (MGA1) is an autosomal recessive disorder caused by the selective intestinal malabsorption of intrinsic factor (IF) and vitamin B12/cobalamin (Cbl) in complex. Most Finnish patients with MGA1 carry the disease-specific P1297L mutation (FM1) in the IF-B12 receptor, cubilin. By site-directed mutagenesis, mammalian expression, and functional comparison of the purified wild-type and FM1 mutant forms of the IF–Cbl-binding cubilin region (CUB domains 5-8, amino acid 928-1386), we have investigated the functional implications of the P1297L mutation. Surface plasmon resonance analysis revealed that the P1297L substitution specifically increases the Kd for IF–Cbl binding several-fold, largely by decreasing the association rate constant. In agreement with the binding data, the wild-type protein, but not the FM1 mutant protein, potently inhibits 37°C uptake of iodine 125–IF–Cbl in cubilin-expressing epithelial cells. In conclusion, the data presented show a substantial loss in affinity of the FM1 mutant form of the IF–Cbl binding region of cubilin. This now explains the malabsorption of Cbl and Cbl-dependent anemia in MGA1 patients with the FM1 mutation.
NFAT-controlled expression of GFP permits visualization and isolation of antigen-stimulated primary human T cells
Authors: Hooijberg Erik , Bakker Arjen Q. , Ruizendaal Janneke J. , Spits Hergen
Published: October 2019

We have developed a new method that allows detection and isolation of viable, antigen-specific, human T cells from a heterogeneous pool of T cells. We have engineered a self-inactivating retroviral vector containing multiple (3 or 6) nuclear factor of activated T-cell (NFAT)-binding sites, followed by the minimal IL2 promoter and the reporter gene GFP. Jurkat cells, primary T-cell blasts, and T-cell clones were transduced with high efficiency (20%-40%). Stimulation of the transduced cells with phorbol myristate acetate (PMA) and ionomycin resulted in a high level expression of GFP that was maximal after 12 to 14 hours and remained stable for another 12 hours. Activation of T cells carrying the construct containing 6 NFAT-binding sites resulted in the highest mean fluorescence intensity. Cyclosporin-A and FK506 were able to block the activation-dependent GFP expression. Activation of transduced T-cell blasts with the superantigen staphylococcal enterotoxin B or of transduced antigen-specific T-cell clones with cognate antigen resulted in GFP expression. After an overnight stimulation of a heterogeneous T-cell bulk culture with an HLA mismatched stimulator cell (JY), the GFP expressing cells were cloned. As expected, the cloning frequency of the antigen-specific GFP+ cells was considerably higher than that of the total T-cell population. Most of the T-cell clones were either cytolytic, or proliferative toward JY stimulator cells. Interestingly, we also isolated T-cell clones that were noncytolytic and nonproliferative toward JY cells, but specifically up-regulated GFP after an overnight stimulation with JY.