bluuebunny/crossref_metadata_2025 huggingface dataset:
Inhibition of Granulocyte-Macrophage Colony-Stimulating Factor Prevents Dissemination and Induces Remission of Juvenile Myelomonocytic Leukemia in Engrafted Immunodeficient Mice
Authors: Iversen Per O. , Lewis Ian D. , Turczynowicz Suzanne , Hasle Henrik , Niemeyer Charlotte , Schmiegelow Kjeld , Bastiras Stan , Biondi Andrea , Hughes Timothy P. , Lopez Angel F.
Published: October 2019

Granulocyte-macrophage colony-stimulating factor (GM-CSF ) and tumor necrosis factor α (TNFα) have been implicated in the pathogenesis of the fatal childhood disease termed juvenile myelomonocytic leukemia (JMML). We used a severe combined immunodeficient/nonobese diabetic (SCID/NOD) mouse model of JMML and examined the effect of inhibiting these cytokines in vivo with the human GM-CSF antagonist and apoptotic agent E21R and the anti-TNFα monoclonal antibody (MoAb) cA2 on JMML cell growth and dissemination in vivo. We show here that JMML cells repopulated to high levels in the absence of exogeneous growth factors. Administration of E21R at the time of transplantation or 4 weeks after profoundly reduced JMML cell load in the mouse bone marrow. In contrast, MoAb cA2 had no effect on its own, but synergized with E21R in virtually eliminating JMML cells from the mouse bone marrow. In the spleen and peripheral blood, E21R eliminated JMML cells, while MoAb cA2 had no effect. Importantly, studies of mice engrafted simultaneously with cells from both normal donors and from JMML patients showed that E21R preferentially eliminated leukemic cells. This is the first time a specific GM-CSF inhibitor has been used in vivo, and the results suggest that GM-CSF plays a major role in the pathogenesis of JMML. E21R might offer a novel and specific approach for the treatment of this aggressive leukemia in man.
Heteroplasmic Point Mutations of Mitochondrial DNA Affecting Subunit I of Cytochrome c Oxidase in Two Patients With Acquired Idiopathic Sideroblastic Anemia
Authors: Gattermann Norbert , Retzlaff Stefan , Wang Yan-Ling , Hofhaus Götz , Heinisch Jürgen , Aul Carlo , Schneider Wolfgang
Published: October 2019

Mitochondrial iron overload in acquired idiopathic sideroblastic anemia (AISA) may be attributable to mutations of mitochondrial DNA (mtDNA), because these can cause respiratory chain dysfunction, thereby impairing reduction of ferric iron (Fe3+) to ferrous iron (Fe2+). The reduced form of iron is essential to the last step of mitochondrial heme biosynthesis. It is not yet understood to which part of the respiratory chain the reduction of ferric iron is linked. In two patients with AISA we identified point mutations of mtDNA affecting the same transmembrane helix within subunit I of cytochrome c oxidase (COX I; ie, complex IV of the respiratory chain). The mutations were detected by restriction fragment length polymorphism analysis and temperature gradient gel electrophoresis. One of the mutations involves a T → C transition in nucleotide position 6742, causing an amino acid change from methionine to threonine. The other mutation is a T → C transition at nt 6721, changing isoleucine to threonine. Both amino acids are highly conserved in a wide range of species. Both mutations are heteroplasmic, ie, they establish a mixture of normal and mutated mitochondrial genomes, which is typical of disorders of mtDNA. The mutations were present in bone marrow and whole blood samples, in isolated platelets, and in granulocytes, but appeared to be absent from T and B lymphocytes purified by immunomagnetic bead separation. They were not detected in buccal mucosa cells obtained by mouthwashes and in cultured skin fibroblasts examined in one of the patients. In both patients, this pattern of involvement suggests that the mtDNA mutation occurred in a self-renewing bone marrow stem cell with myeloid determination. Identification of two point mutations with very similar location suggests that cytochrome c oxidase plays an important role in the pathogenesis of AISA. COX may be the physiologic site of iron reduction and transport through the inner mitochondrial membrane.
Ex Vivo Generation of Human Anti–Pre-B Leukemia-Specific Autologous Cytolytic T Cells
Authors: Cardoso Angelo A. , Seamon Mark J. , Afonso Hernani M. , Ghia Paolo , Boussiotis Vassiliki A. , Freeman Gordon J. , Gribben John G. , Sallan Stephen E. , Nadler Lee M.
Published: October 2019

In contrast to other neoplasms, antigen-specific autologous cytolytic T cells have not been detected in patients with human pre-B–cell leukemias. The absence of efficient B7 family (B7-1/CD80; B7-2/CD86) -mediated costimulation has been shown to be a major defect in tumor cells' capacity to function as antigen-presenting cells. We show here the generation of autologous anti–pre-B–cell leukemia-specific cytolytic T-cell lines from the marrows of 10 of 15 patients with pre-B–cell malignancies. T-cell costimulation via CD28 is an absolute requirement for the generation of these autologous cytolytic T cells (CTL). Although costimulation could be delivered by either bystander B7 transfectants or professional antigen-presenting cells (indirect costimulation), optimal priming and CTL expansion required that the costimulatory signal was expressed by the tumor cell (direct costimulation). These anti–pre-B–cell leukemia-specific CTL lysed both unstimulated and CD40-stimulated tumor cells from each patient studied but did not lyse either K562 or CD40-stimulated allogeneic B cells. Cytolysis was mediated by the induction of tumor cell apoptosis by CD8+ T cells via the perforin-granzyme pathway. Although we were able to generate anti–leukemia-specific CTL from the bone marrow, we were unable to generate such CTL from the peripheral blood of these patients. These studies show that antigen-specific CTL can be generated from the bone marrow of patients with pre-B–cell leukemias and these findings should facilitate the design of adoptive T-cell–mediated immunotherapy trials for the treatment of patients with B-cell precursor malignancies.
In Vivo Effects of Flt3/Flk2 Ligand on Mobilization of Hematopoietic Progenitors in Primates and Potent Synergistic Enhancement With Granulocyte Colony-Stimulating Factor
Authors: Papayannopoulou Thalia , Nakamoto Betty , Andrews Robert G. , Lyman Stewart D. , Lee Minako Y.
Published: October 2019

The Flt3 receptor is expressed in primitive hematopoietic cells and its ligand exerts proliferative effects on these cells in vitro in synergy with other cytokines. To expand on the functional properties of Flt3 ligand (FL) in vivo we treated nonhuman primates with FL and tested its ability to mobilize stem/progenitor cells when given alone or in combination with granulocyte colony-stimulating factor (G-CSF ) treatment. FL alone (200 μg/kg/day) mobilizes progenitors with slow kinetics and with a peak effect at the end of 2 weeks of treatment. The spectrum of mobilized progenitors includes myeloid, lymphoid, megakaryocytic, and osteoclastogenic but a low proportion of burst-forming unit (BFU)e. Bone marrow (BM) studies before and during the treatment suggested that proliferative effects in BM may have preceded effects on peripheral blood mobilization. To assess the synergy of FL with G-CSF in mobilization of progenitors we used two schemes: one in which G-CSF was used for the last 5 days of a 12-day treatment with FL; the other in which both cytokines were given concurrently for 5 days only (FL, 200 μg/kg; G-CSF, 100 μg/kg). Both schemes yielded much higher progenitor mobilization levels (peak levels of colony-forming cells [CFSs] 41,000 to 95,000/mL blood) than observed with either FL (CFC 4,600 to 7,300/mL) or G-CSF (8,405 ± 3,024/mL) used alone at the same doses. Furthermore, there was a progressive and significant expansion of progenitors in vitro during 2 weeks in suspension cultures of mononuclear cells or of CD34+ cells only in the animal with the combined treatment. Likewise, substantial mobilization of osteoclastogenic progenitors was documented only with the combined treatment. Given the functional properties of FL, its synergistic mobilization with G-CSF, and its anticipated good tolerance (because of the absence of an effect on mast cell activation), a clinical use is projected for this cytokine in peripheral blood transplantation settings, as well as in experiments with ex vivo gene transfer.
Differential Maintenance of Primitive Human SCID-Repopulating Cells, Clonogenic Progenitors, and Long-Term Culture-Initiating Cells After Incubation on Human Bone Marrow Stromal Cells
Authors: Gan Olga I. , Murdoch Barbara , Larochelle Andre , Dick John E.
Published: October 2019

Many experimental and clinical protocols are being developed that involve ex vivo culture of human hematopoietic cells on stroma or in the presence of cytokines. However, the effect of these manipulations on primitive hematopoietic cells is not known. Our severe combined immune-deficient mouse (SCID)-repopulating cell (SRC) assay detects primitive human hematopoietic cells based on their ability to repopulate the bone marrow (BM) of immune-deficient non-obese diabetic/SCID (NOD/SCID) mice. We have examined here the maintenance of SRC, colony-forming cells (CFC), and long-term culture-initiating cells (LTC-IC) during coculture of adult human BM or umbilical cord blood (CB) cells with allogeneic human stroma. Transplantation of cultured cells in equivalent doses as fresh cells resulted in lower levels of human cell engraftment after 1 and 2 weeks of culture for BM and CB, respectively. Similar results were obtained using CD34+-enriched CB cells. By limiting dilution analysis, the frequency of SRC in BM declined sixfold after 1 week of culture. In contrast to the loss of SRC as measured by reduced repopulating capacity, the transplanted inocula of cultured cells frequently contained equal or higher numbers of CFC and LTC-IC compared with the inocula of fresh cells. The differential maintenance of CFC/LTC-IC and SRC suggests that SRC are biologically distinct from the majority of these in vitro progenitors. This report demonstrates the importance of the SRC assay in the development of ex vivo conditions that will allow maintenance of primitive human hematopoietic cells with repopulating capacity.
A Three Amino Acid Deletion in Glycoprotein IIIa Is Responsible for Type I Glanzmann's Thrombasthenia: Importance of Residues Ile325Pro326Gly327 for β3 Integrin Subunit Association
Authors: Morel-Kopp Marie-Christine , Kaplan Cécile , Proulle Valérie , Jallu Vincent , Melchior Chantal , Peyruchaud Olivier , Aurousseau Marie-Hélène , Kieffer Nelly
Published: October 2019

Glanzmann's thrombasthenia (GT) is a recessive autosomal bleeding disorder characterized by abnormal platelet aggregation due to a qualitative or quantitative defect of the glycoprotein (GP) IIb-IIIa complex (integrin αIIbβ3). We describe a new mutation in the GPIIIa gene responsible for type I GT in a consanguineous Algerian family. A discordance between phenotyping and genotyping of the GPIIIa-related HPA-1 platelet alloantigen system in three family members heterozygous for the disease suggested a genetic defect in the GPIIIa gene and a normal GPIIb gene. Sequence analysis of amplified genomic DNA fragments showed a 6-bp deletion in exon 7 of the GPIIIa gene resulting in the amino acid deletion/substitution (Ile325Pro326Gly327 → Met) and creating a new BspHI restriction site. Expression of the mutated integrin β3 subunit cDNA in Chinese hamster ovary cells showed that the cDNA gene was transcribed into a full-length β3 protein with an apparent molecular weight identical to wild-type β3 and accumulated as a single-chain molecule in the cell cytoplasm. The absence of heterodimeric complex formation of the mutant β3 protein with endogeneous αv was shown by immunoprecipitation experiments, intracellular immunofluorescent labeling, and a semiquantitative enzyme-linked immunosorbent assay using the αvβ3 complex-specific monoclonal antibodies LM609 and 23C6. Substitution of the methionine residue by a proline, present at position 326 of wild-type β3, did not restore the ability of the recombinant mutant β3 protein to associate with αv, suggesting that the Ile-Pro-Gly motif is located in a β3 domain important for integrin subunit interaction. The association of a BspHI restriction site with this newly identified mutation has allowed allele-specific restriction analysis of Algerian GT individuals and the identification of two new unrelated type I patients exhibiting the same mutation, suggesting that the described mutation might be significant in this population and that BspHI restriction analysis will provide a useful screening assay for antenatal diagnosis and genetic counselling.
Signaling Pathways Regulating CD44-Dependent Cytolysis in Natural Killer Cells
Authors: Sconocchia Giuseppe , Titus Julie A. , Segal David M.
Published: October 2019

CD44 is a cytotoxic triggering molecule on activated, but not fresh natural killer (NK) cells. In the current study, metabolic pathways used in CD44-directed lysis (CD44DL) were examined using activated human NK cells as effectors. We found that CD44 expressed by activated NK cells was indistinguishable in isoform and molecular weight from CD44 on unactivated cells. However, de novo protein expression was required for the induction of CD44DL, suggesting that activated NK cells contain proteins not present in fresh NK cells that couple CD44 to the lytic machinery. Concanimycin A, a selective inhibitor of perforin-based cytolysis, totally blocked CD44DL, natural cytototoxicity, and antibody-dependent cell-mediated cytolysis (ADCC). Moreover, studies in which kinase inhibitors were added during the effector phase of lysis indicated that protein-tyrosine and ser/thr kinases were required for all three cytolytic activities and that protein kinase C played a nonessential role in lysis. By contrast, wortmannin totally inhibited CD44DL, but failed to block natural cytotoxicity and only partially blocked ADCC, suggesting that phosphatidylinositol 3-kinase (PI 3-kinase) is required at an early, receptor-specific stage of CD44DL. Finally, cytochalasin B enhanced CD44DL, but not ADCC, indicating that CD44DL is modulated by actin polymerization. Taken together, our data suggest that CD44 in NK cells interacts with proteins induced during interleukin-2 activation in a triggering pathway that induces perforin release, requires PI 3-kinase, and is modulated by the cytoskeleton.
Hereditary Hyperferritinemia-Cataract Syndrome: Relationship Between Phenotypes and Specific Mutations in the Iron-Responsive Element of Ferritin Light-Chain mRNA
Authors: Cazzola Mario , Bergamaschi Gaetano , Tonon Laura , Arbustini Eloisa , Grasso Maurizia , Vercesi Elena , Barosi Giovanni , Bianchi Paolo Emilio , Cairo Gaetano , Arosio Paolo
Published: October 2019

Recent reports have described families in whom a combination of elevated serum ferritin not related to iron overload and congenital nuclear cataract is transmitted as an autosomal dominant trait. We have studied the molecular pathogenesis of hyperferritinemia in two families showing different phenotypic expression of this new genetic disorder. Serum ferritin levels ranged from 950 to 1,890 μg/L in affected individuals from family 1, and from 366 to 635 μg/L in those from family 2. Cataract was clinically manifested in family 1 and asymptomatic in family 2. By using monoclonal antibodies specific for the H and L ferritin subunits, serum ferritin was found to be essentially L type in both normal and affected individuals. The latter also showed normal amounts of H-type ferritin in circulating mononuclear cells; on the contrary, L-type ferritin contents were 13 times normal in family 1 and five times normal in family 2 on average. Serum ferritin was glycosylated in both normal and affected individuals. There was a close relationship between mononuclear cell L-type ferritin content and serum ferritin concentration (r = 0.95, P < .00001), suggesting that the excess production of ferritin in cells was directly responsible for the hyperferritinemia. The dysregulated L-subunit synthesis was found to result from different point mutations in a noncoding sequence of genomic L-subunit DNA, which behaves as an mRNA cis-acting element known as iron regulatory element (IRE). Affected individuals from family 1 were heterozygous for a point mutation (a single G to A change) in the highly conserved, three-nucleotide motif forming the IRE bulge. Affected members from family 2 were heterozygous for a double point mutation in the IRE lower stem. Using a gel retardation assay, the observed molecular lesions were shown to variably reduce the IRE affinity for an iron regulatory protein (IRP), which inhibits ferritin mRNA translation. The direct relationship between the degree of hyperferritinemia and severity of cataract suggests that this latter is the consequence of excessive ferritin production within the lens fibers. These findings provide strong evidence that serum ferritin is a byproduct of intracellular ferritin synthesis and that the L-subunit gene on chromosome 19 is the source of glycosylated serum ferritin. From a practical standpoint, this new genetic disorder should be taken into account by clinicians when facing a high serum ferritin in an apparently healthy person.
The Pattern of Interleukin-1β (IL-1β) and Its Modulating Agents IL-1 Receptor Antagonist and IL-1 Soluble Receptor Type II in Acute Meningococcal Infections
Authors: van Deuren Marcel , van der Ven-Jongekrijg Johanna , Vannier Edouard , van Dalen Roelof , Pesman Gerard , Bartelink Anton K.M. , Dinarello Charles A. , W.M. van der Meer Jos
Published: October 2019

Interleukin-1β (IL-1β) is considered an important mediator in the pathogenesis of septic shock or bacterial meningitis. Its activity is specifically modulated by IL-1 receptor antagonist (IL-1Ra) and IL-1 soluble receptor type II (IL-1sRII). We now describe the time-course of IL-1β and these modulating agents in 59 patients with acute meningococcal infections, the prototype human disease of acute endotoxin exposure. Plasma IL-1β was increased only in severe shock and normalized within 12 to 24 hours, indicating that patients were admitted in an early stage of cytokine activation. Increased IL-1β values in cerebrospinal fluid (CSF ) were confined to patients with meningitis. Plasma IL-1Ra was elevated in both shock and nonshock patients, extremely high values being measured in severe shock. High concentrations of IL-1Ra in CSF were found in meningitis. Plasma IL-1Ra peaked shortly after IL-1β and decreased steeply in 1 to 2 days, followed by sustained moderately elevated levels in shock patients. Interestingly, IL-1sRII showed a completely different pattern. At admission, both nonshock and shock patients manifested a similar moderate increase of plasma IL-1sRII. However, during recovery plasma IL-1sRII further increased reaching maximal concentrations 3 to 5 days after admission, 1 to 2 days after normalization of IL-1Ra. In shock patients this increase was more prominent than in nonshock patients. It is hypothesized that this increase in plasma IL-1sRII can be explained by a synergistic effect of dexamethasone and endotoxin. A second interesting observation was that, unlike the pattern in plasma, IL-1sRII levels in CSF paralleled those of IL-1β and IL-1Ra. This suggests different modulation of IL-1β activity in the subarachnoid space and the plasma compartment. We conclude that: (1) During the early stage of meningococcal infections IL-1Ra modulates IL-1 activity, whereas during recovery IL-1sRII may be more important. (2) Modulation in CSF and in the plasma compartment are differentially regulated.
BCL2 Overexpression Associated With Chromosomal Amplification in Diffuse Large B-Cell Lymphoma
Authors: Monni Outi , Joensuu Heikki , Franssila Kaarle , Klefstrom Juha , Alitalo Kari , Knuutila Sakari
Published: October 2019

Gene activation by translocation between an oncogene and an immunoglobulin heavy-chain gene, which leads to increased expression of the oncoprotein, is a well-known mechanism in the genesis of B-cell lymphomas. In contrast, the role of gene amplification in activation of oncogenes in non-Hodgkin's lymphomas is poorly characterized. To study the BCL2 amplification we performed comparative genomic hybridization (CGH), Southern blot hybridization, Western analysis, immunohistochemistry, metaphase fluorescence in situ hybridization, and chromosome analysis on 26 cases of diffuse large B-cell lymphoma (large noncleaved cell lymphoma). The gain or high-level amplification of 18q was found in eight tumors (31%) by CGH, and Southern analysis revealed BCL2 amplification in these cases, but not in the cases with normal chromosome 18 or t(14; 18)(q32; q21). Western immunoblot analysis and immunohistochemistry revealed a high-level expression of BCL2 protein in the cases with BCL2 amplification and t(14; 18)(q32; q21). However, translocation (14; 18)(q32; q21) was not detected in any of the cases with BCL2 amplification. Therefore, our results suggest that amplification of the BCL2 gene is an important mechanism for BCL2 protein overexpression in diffuse large B-cell lymphoma.
Human Herpesvirus 6 (HHV-6)–Positive Burkitt's Lymphoma: Establishment of a Novel Cell Line Infected With HHV-6
Authors: Bandobashi Kentaro , Daibata Masanori , Kamioka Mikio , Tanaka Yuji , Kubonishi Ichiro , Taguchi Hirokuni , Ohtsuki Yuji , Miyoshi Isao
Published: October 2019

Human herpesvirus 6 (HHV-6) DNA has been detected in several human lymphoproliferative disorders. We report a case of HHV-6–infected Burkitt's lymphoma, from which a cell line, designated Katata, has been established. Katata cells had an immature B-cell phenotype with an L3 morphology and carried a t(8; 14)(q24; q32) chromosomal abnormality. The HHV-6 DNA sequences were detected in both the patient's tumor cells and Katata cell line by polymerase chain reaction using three sets of primers that target different regions of HHV-6 DNA. The presence of HHV-6 DNA in Katata cells was also shown by Southern blot hybridization with the BamHI fragment of HHV-6. It is likely that the virus is in a latent state, since (1) virion-associated protein was not expressed in Katata cells, (2) transcriptional level of the immediate-early gene was very low, and (3) no viral particles were observed by electron microscopy. Katata cells were highly tumorigenic in nude mice and the tumor cells also contained HHV-6 DNA. We have successfully obtained several clonal lines by allowing the cells to form colonies in soft agarose and by the limiting dilution method. HHV-6 DNA was detectable in all 13 clones analyzed, suggesting that virtually all Katata cells are infected with HHV-6. This is the first report of a case of HHV-6+ Burkitt's lymphoma in the absence of Epstein-Barr virus. Furthermore, there has been no report of lymphoma cell lines that are persistently and nonproductively infected with HHV-6. The Katata Burkitt's lymphoma cell line, therefore, would provide a useful tool for studies of the mechanisms of HHV-6 latency and reactivation.
Expression and Intracellular Localization of the Human N-acetylmuramyl-L-alanine Amidase, a Bacterial Cell Wall–Degrading Enzyme
Authors: Hoijer M.A. , Melief M.J. , Calafat J. , Roos D. , van den Beemd R.W.M. , van Dongen J.J.M. , Hazenberg M.P.
Published: October 2019

N-acetylmuramyl-L-alanine amidase (NAMLAA) specifically degrades peptidoglycan, which is a major component of bacterial cell walls with strong inflammatory properties. For instance, peptidoglycan is capable of stimulating peripheral blood cells to release pro-inflammatory cytokines and is capable of inducing chronic arthritis in an animal model. In a previous study we found that degradation of peptidoglycan by purified NAMLAA reduced its inflammatory effects. To determine where NAMLAA is located in tissues, monoclonal antibodies against purified NAMLAA were produced for use in immunohistochemistry, immunoelectron microscopy, flow cytometric analysis, and Western blotting. The immunohistochemical studies showed NAMLAA-positive cells in human spleen, liver, arthritic synovial tissues, and lymph nodes. In flow cytometric studies of blood and bone marrow, neutrophilic and eosinophilic granulocytes proved to be positive. Monocytes were negative, although they do contain lysozyme, the other important peptidoglycan-degrading enzyme. However, mature macrophages obtained by bronchoalveolar lavage and subsequent selection based on auto-fluorescence did possess NAMLAA. In immunocytochemical staining of blood smears, thrombocytes were also positive for NAMLAA. Western blot analysis and immunoelectron microscopy of neutrophils and eosinophils showed that NAMLAA is located in azurophilic granules of neutrophils and in secretory vesicles and crystalloid-containing granules of eosinophils. Flow cytometric analysis of blood and bone marrow from different French-American-British–classified acute myeloid leukemia (AML) patients showed that AML-M2 myeloblasts were the first in the granulocyte maturation lineage that were positive for NAMLAA. The more immature AML, such as AML-M0 and AML-M1, did not express NAMLAA. CD15- and CD13-negative megakaryoblasts, corresponding to AML-M7, were also positive for NAMLAA. The expression pattern of NAMLAA in the myeloid lineage suggests that the monoclonal antibody AAA4, recognizing NAMLAA, is useful for discrimination between AML in the monocyte lineage and in the granulocyte lineage.
FcγRII (CD32) Is Linked to Apoptotic Pathways in Murine Granulocyte Precursors and Mature Eosinophils
Authors: de Andrés Belen , Mueller Allen L. , Blum Arthur , Weinstock Joel , Verbeek Sjef , Sandor Matyas , Lynch Richard G.
Published: October 2019

Murine granulocytes and precursors express low-affinity IgG Fc receptors (FcγR). We investigated the effects of FcγR ligation on the development of eosinophils in cultures of normal murine bone marrow. Eosinophilopoiesis was induced by culture of bone marrow cells in the presence of cytokines (granulocyte-macrophage colony-stimulating factor [GM-CSF], interleukin-3 [IL-3], and IL-5). Addition to the cultures of 2.4G2, a rat monoclonal antibody (mAb) that reacts with FcγRII (CD32) and FcγRIII (CD16), induced granulocyte apoptosis within 24 hours. Granulocytes in cultures that contained 2.4G2 showed chromatin condensation, binding of Annexin-V, and fas induction, and by electron microscopy, apoptosis was most commonly observed in cells of the eosinophil lineage. Since murine granulocytes can express both FcγRII (CD32) and FcγRIII (CD16), we investigated the effect of 2.4G2 on cultures of bone marrow obtained from FcγRIII (CD16) gene–disrupted mice and found that the apoptosis induced with 2.4G2 was CD16-independent. Studies with bone marrow cultures from B6MLR-lpr/lpr and C3H/HEJ-gld/gld mice established that the FcγRII (CD32)-triggered apoptosis was fas-fasL–dependent. When mature eosinophils isolated from hepatic granulomas of Schistosoma mansoni–infected mice were cultured in cytokines in the presence of 2.4G2, the eosinophils underwent apoptosis within 24 hours. These findings identify a previously unknown linkage between FcγR on eosinophils and fas-mediated apoptosis, a connection that could be relevant to mechanisms by which eosinophils mediate tissue injury and antibody-dependent cellular cytotoxicity reactions.
Inactivation of the Fanconi Anemia Group C Gene Augments Interferon-γ–Induced Apoptotic Responses in Hematopoietic Cells
Authors: Rathbun R. Keaney , Faulkner Gregory R. , Ostroski Marika H. , Christianson Tracy A. , Hughes Grant , Jones Gary , Cahn Robert , Maziarz Richard , Royle Gordon , Keeble Winifred , Heinrich Michael C. , Grompe Markus , Tower Paula A. , Bagby Grover C.
Published: October 2019

Hematopoietic progenitor cells (HPC) from mice nullizygous at the Fanconi anemia (FA) group C locus (FAC −/−) are hypersensitive to the mitotic inhibitory effects of interferon (IFN-γ). We tested the hypothesis that HPC from the bone marrow of Fanconi group C children are similarly hypersensitive and that the fas pathway is involved in affecting programmed cell death in response to low doses of IFN-γ. In normal human and murine HPC, IFN-γ primed the fas pathway and induced both fas and interferon response factor-1 (IRF-1) gene expression. These IFN-γ-induced apoptotic responses in HPC from the marrow of a child with FA of the C group (FA-C) and in FAC −/− mice occurred at significantly lower IFN doses (by an order of magnitude) than did the apoptotic responses of normal HPC. Treatment of FA-C CD34+ cells with low doses of recombinant IFN-γ, inhibited growth of colony forming unit granulocyte-macrophage and burst-forming unit erythroid, while treatment with blocking antibodies to fas augmented clonal growth and abrogated the clonal inhibitory effect of IFN-γ. Transfer of the normal FAC gene into FA-C B-cell lines prevented mitomycin C–induced apoptosis, but did not suppress fas expression or inhibit the primed fas pathway. However, the kinetics of Stat1-phosphate decay in IFN-γ–treated cells was prolonged in mutant cells and was normalized by transduction of the normal FAC gene. Therefore, the normal FAC protein serves, in part, to modulate IFN-γ signals. HPC bearing inactivating mutations of FAC fail to normally modulate IFN-γ signals and, as a result, undergo apoptosis executed through the fas pathway.
Kinetics of Central Nervous System Microglial and Macrophage Engraftment: Analysis Using a Transgenic Bone Marrow Transplantation Model
Authors: Kennedy David W. , Abkowitz Janis L.
Published: October 2019

To determine the kinetics of tissue macrophage and microglial engraftment after bone marrow (BM) transplantation, we have developed a model using the ROSA 26 mouse. Transplanted ROSA 26 cells can be precisely identified in recipient animals because they constitutively express β-galactosidase (β-gal) and neomycin resistance. B6/129 F2 mice were irradiated and transplanted with BM from ROSA 26 donors and their tissues (spleen, marrow, brain, liver, and lung) examined at various time points to determine the kinetics of engraftment. Frozen sections from transplanted animals were stained histochemically for β-gal to identify donor cells. At 1, 2, 6, and 12 months posttransplantation, 98% to 100% of granulocyte-macrophage colonies were of donor (ROSA 26) origin determined by β-gal staining and by neomycin resistance. Splenic monocytes/macrophages were 89% donor origin by 1 month confirming quick and complete engraftment of hematopoietic tissues. At this time, only rare ROSA 26 tissue macrophages or microglia were observed. Alveolar macrophage engraftment was evident by 2 months and had increased to 61% of total tissue macrophages at 1 year posttransplantation. The kinetics of liver Kupffer cell engraftment were similar to those seen in the lung. However, donor microglial engraftment remained only 23% of total microglia at 6 months and increased to only 30% by 1 year. Also, donor microglia were predominantly seen at perivascular and leptomeningeal, and not parenchymal, sites. The data show that microglia derive from BM precursors but turn over at a significantly slower rate than other tissue macrophages. No clinical or histological graft-versus-host disease was observed in the recipients of ROSA 26 BM. These kinetics may impact strategies for the gene therapy of lysosomal storage diseases. Because individual donor cells can be identified in situ, the ROSA 26 model should have many applications in transplantation biology including studies of homing and differentiation.
Development of a Candidate HLA A*0201 Restricted Peptide-Based Vaccine Against Human Cytomegalovirus Infection
Authors: Diamond Don J. , York Joanne , Sun Ji-Yao , Wright Christine L. , Forman Stephen J.
Published: October 2019

The development of a protective cellular immune response against human cytomegalovirus (HCMV) is the most important determinant of recovery from HCMV infection after allogeneic bone marrow transplantation (BMT). The ultimate aim of our study is to develop an antigen-specific and peptide-based vaccine strategy against HCMV in the setting of BMT. Toward this end we have studied the cellular immune response against the immunodominant matrix protein pp65 of HCMV. Using an HLA A*0201-restricted T-cell clone reactive against pp65 from peripheral blood from a seropositive individual, we have mapped the position of the cytolytic T lymphocyte (CTL) epitope from HCMV pp65 to an 84-amino acid segment. Of the four peptides which best fit the HLA A*0201 motif in that region, one nonamer sensitized an autologous Epstein-Barr virus immortalized lymphocyte cell line for lysis. In vitro immunization of PBMC from HLA A*0201 and HCMV seropositive volunteers using the defined nonamer peptide stimulated significant recognition of HCMV infected or peptide-sensitized fibroblasts. Similarly, HLA A*0201 transgenic mice immunized with the nonamer peptide developed CTL that recognize both the immunizing peptide and endogenously processed pp65 in an HLA A*0201 restricted manner. Lipid modification of the amino terminus of the nonamer peptide resulted in its ability to stimulate immune respones without the use of adjuvant. This demonstration of a vaccine function of the nonamer peptide without adjuvant suggests its potential for use in an immunization trial of BMT donors to induce protective CTLs in patients undergoing allogeneic BMT.
c-Cbl Is Tyrosine-Phosphorylated by Interleukin-4 and Enhances Mitogenic and Survival Signals of Interleukin-4 Receptor by Linking With the Phosphatidylinositol 3′-Kinase Pathway
Authors: Ueno Hiroo , Sasaki Ko , Honda Hiroaki , Nakamoto Tetsuya , Yamagata Tetsuya , Miyagawa Kiyoshi , Mitani Kinuko , Yazaki Yoshio , Hirai Hisamaru
Published: October 2019

Interleukin-4 (IL-4) is a cytokine that induces both proliferation and differentiation and suppresses apoptosis of B cells. Although IL-4 has been shown to activate the phosphatidylinositol 3′ (PI3)-kinase pathway, the role of PI3 kinase in the IL-4 receptor (IL-4R) signaling remains unclear. In this study, we demonstrated that c-Cbl proto-oncogene product is inducibly phosphorylated on tyrosine residues and is associated with the p85 subunit of PI3-kinase by IL-4 stimulation. Overexpression of c-Cbl enhances the PI3-kinase activity and, at the same time, mitogenic activity and survival of cells in the presence of IL-4. However, these effects of c-Cbl were abolished by wortmannin, a specific inhibitor for the PI3 kinase pathway, or by a point mutation at tyrosine 731 of c-Cbl, which is a major binding site for p85. These results indicate that c-Cbl plays a role in linking IL-4R with the PI3 kinase pathway and thus enhancing the mitogenic and survival signals.
Prothrombin G20210A Mutant Genotype Is a Risk Factor for Cerebrovascular Ischemic Disease in Young Patients
Authors: De Stefano Valerio , Chiusolo Patrizia , Paciaroni Katia , Casorelli Ida , Rossi Elena , Molinari Marco , Servidei Serenella , Tonali Pietro A. , Leone Giuseppe
Published: October 2019

The factor II G20210A mutation is a recently identified congenital risk factor for venous thrombosis. Its role in artery disease is still undefined. We investigated 72 patients (35 male and 37 female) with documented ischemic stroke occurred before 50 years of age and without risk factors such as diabetes, hypertension, and hyperlipidemia; 198 thrombosis-free individuals were investigated as the control group. We found 7 heterozygotes (9.7%) and 2 homozygotes (2.7%) for the mutant factor II allele among the patients and 5 heterozygotes (2.5%) among the controls; the mutant factor II allele frequency in the patient group (7.6%, 95% confidence interval [CI], 3.3 to 11.9) was significantly higher than in the controls (1.2%; 95% CI, 0.1 to 2.3;P = .0001). The prevalence of other investigated mutant alleles (factor V G1691A, methylenetetrahydrofolate reductase C677T) did not significantly differ between the two groups. The odds ratio for ischemic stroke associated with the carriership of the mutant factor II allele (both heterozygous and homozygous genotypes) was 5.1 (95% CI, 1.6 to 16.3). Heterozygous genotype was associated with a 3.8-fold increased risk for cerebral ischemia (95% CI, 1.1 to 13.1); in particular, assuming an expected prevalence of homozygotes in the general population of 1.6 to 10,000 according to the Hardy-Weinberg equilibrium, the risk associated with the homozygous genotype was estimated exceedingly high, being increased 208-fold.
Secondary Mutation Maintains the Transformed State in BaF3 Cells With Inducible BCR/ABL Expression
Authors: Klucher Kevin M. , Lopez David V. , Daley George Q.
Published: October 2019

The BCR/ABL gene product of the Philadelphia (Ph) chromosome induces chronic myelogenous leukemia (CML). We generated a hematopoietic cell line, TonB210.1, with tetracycline-dependent BCR/ABL expression to investigate the pathways by which BCR/ABL transforms cells. TonB210.1 demonstrates conditional growth factor independence in tissue culture and rapidly forms tumors in mice fed the tetracycline analog doxycycline. The tumors regress completely upon doxycycline withdrawal, but ultimately reform in all animals. After a long latency, tumors also develop in animals never exposed to doxycycline. Subclones of TonB210.1 established from doxycycline-independent tumors demonstrate distinct mechanisms of transformation. Most subclones manifest increased basal levels of BCR/ABL expression; some have lost the capacity to augment expression upon induction, whereas others remain inducible. More interestingly, some subclones maintain tight conditional expression of BCR/ABL and are therefore transformed by secondary mechanisms that no longer require BCR/ABL expression. These subclones show constitutive phosphorylation of the STAT5 protein, suggesting that activating mutations have occurred upstream in the signaling pathway to STAT5. The tight conditional expression of BCR/ABL in the TonB210.1 cell line affords the opportunity to study several interesting aspects of the biology of BCR/ABL, including activation of critical signaling pathways and transcriptional programs, and its potential role in genomic instability.
The Related Adhesion Focal Tyrosine Kinase (RAFTK) Is Tyrosine Phosphorylated and Participates in Colony-Stimulating Factor-1/Macrophage Colony-Stimulating Factor Signaling in Monocyte-Macrophages
Authors: Hatch William C. , Ganju Ramesh K. , Hiregowdara Dananagoud , Avraham Shalom , Groopman Jerome E.
Published: October 2019

RAFTK, a novel nonreceptor protein kinase, has been shown to be involved in focal adhesion signal transduction pathways in neuronal PC12 cells, megakaryocytes, platelets, and T cells. Because focal adhesions may modulate cytoskeletal functions and thereby alter phagocytosis, cell migration, and adhesion in monocyte-macrophages, we investigated the role of RAFTK signaling in these cells. RAFTK was abundantly expressed in THP1 monocytic cells as well as in primary alveolar and peripheral blood-derived macrophages. Colony-stimulating factor-1 (CSF-1)/macrophage colony-stimulating factor (M-CSF) stimulation of THP1 cells increased the tyrosine phosphorylation of RAFTK; similar increases in phosphorylation were also detected after lipopolysaccharide stimulation. RAFTK was phosphorylated with similar kinetics in THP1 cells and peripheral blood-derived macrophages. Immunoprecipitation analysis showed associations between RAFTK and the signaling molecule phosphatidylinositol-3 (PI-3) kinase. PI-3 kinase enzyme activity also coprecipitated with the RAFTK antibody, further confirming this association. The CSF-1/M-CSF receptor c-fms and RAFTK appeared to associate in response to CSF-1/M-CSF treatment of THP1 cells. Inhibition of RAFTK by a dominant-negative kinase mutant reduced CSF-1/M-CSF–induced MAPK activity. These data indicate that RAFTK participates in signal transduction pathways mediated by CSF-1/M-CSF, a cytokine that regulates monocyte-macrophage growth and function.
Growth Inhibition of Granulocyte-Macrophage Colony-Forming Cells by Human Cytidine Deaminase Requires the Catalytic Function of the Protein
Authors: Gran Christine , Bøyum Arne , Johansen Rune F. , Løvhaug Dagfinn , Seeberg Erling C.
Published: October 2019

Previous studies have indicated that cytidine deaminase (CDD) is a potent growth inhibitor of granulocyte-macrophage colony-forming cells (GM-CFC). In this study, we have undertaken molecular cloning and purification of recombinant human CDD to elucidate the growth regulatory potential and mechanism behind the growth suppressive effect. The purified protein had a specific activity of 1.35 × 105 U/mg and a Km value of 30 μmol/L. In the GM-CFC assay, the recombinant protein was shown to reduce colony formation to 50% at 16 pmol/L concentration. Similarly, as was observed with CDD derived from granulocyte extract, the effect depended on the presence of thymidine (≥ 4 × 10-5 mol/L). These results imply that CDD is an extremely potent inhibitor of GM-CFC and that no additional factor from the granulocyte extract is required for the growth inhibitory effect. Modification of CDD by truncation from the C-terminal end, or by amino acid substitution of an active site glutamate residue, eliminated both the enzyme activity and the growth regulatory potential of CDD. Furthermore, CDD fromEscherichia coli was found to be even more effective than human CDD in growth suppression of GM-CFC, with 10-fold higher inhibitory activity corresponding to a 10-fold higher enzymatic activity. Taken together, these results show that the catalytic nucleoside deaminating function of the protein is essential for the growth suppressive effect of CDD. Most probably, CDD exerts growth inhibition by depleting the cytidine and deoxycytidine pool required for DNA synthesis, as addition of deoxycytidine monophosphate, which is not a substrate for CDD, neutralizes the inhibiting effect.
Biochemical and Pharmacological Properties of SANORG 34006, a Potent and Long-Acting Synthetic Pentasaccharide
Authors: Herbert J.M. , Hérault J.P. , Bernat A. , van Amsterdam R.G.M. , Lormeau J.C. , Petitou M. , van Boeckel C. , Hoffmann P. , Meuleman D.G.
Published: October 2019

SANORG 34006 is a new sulfated pentasaccharide obtained by chemical synthesis. It is an analog of the “synthetic pentasaccharide” (SR 90107/ ORG 31540) which represents the antithrombin (AT) binding site of heparin. SANORG 34006 showed a higher affinity to human AT than SR 90107/ORG 31540 (kd = 1.4 ± 0.3 v 48 ± 11 nmol/L), and it is a potent and selective catalyst of the inhibitory effect of AT on factor Xa (1,240 ± 15 anti–factor Xa U/mg v850 ± 27 anti-factor Xa U/mg for SR 90107/ORG 31540). In vitro, SANORG 34006 inhibited thrombin generation occurring via both the extrinsic and intrinsic pathway. After intravenous (IV) or subcutaneous (SC) administration to rabbits, SANORG 34006 displayed a long-lasting anti–factor Xa activity and inhibition of thrombin generation (TG) ex vivo. SANORG 34006 was slowly eliminated after IV or SC administration to rats, rabbits, and baboons, showed exceptionally long half-lives (between 9.2 hours in rats and 61.9 hours in baboons), and revealed an SC bioavailability near 100%. SANORG 34006 displayed antithrombotic activity by virtue of its potentiation of the anti–factor Xa activity of AT. It strongly inhibited thrombus formation in experimental models of thromboplastin/stasis-induced venous thrombosis in rats (IV) and rabbits (SC) (ED50values = 40.0 ± 3.4 and 105.0 ± 9.4 nmol/kg, respectively). The duration of its antithrombotic effects closely paralleled the ex vivo anti–factor Xa activity. SANORG 34006 enhanced rt-PA–induced thrombolysis and inhibited accretion of125I-fibrinogen onto a preformed thrombus in the rabbit jugular vein suggesting that concomitant use of SANORG 34006 during rt-PA therapy might be helpful in facilitating thrombolysis and preventing fibrin accretion onto the thrombus under lysis. Contrary to standard heparin, SANORG 34006 did not enhance bleeding in a rabbit ear incision model at a dose that equals 10 times the antithrombotic ED50 in this species and, therefore, exhibited a favorable therapeutic index. We suggest that SANORG 34006 is a promising compound in the treatment and prevention of various thrombotic diseases.
Viral Superantigen-Induced Negative Selection of TCR Transgenic CD4+ CD8+ Thymocytes Depends on Activation, but not Proliferation
Authors: Ferrero Isabel , Anjuère Fabienne , Azcoitia Iñigo , Renno Toufic , MacDonald H. Robson , Ardavı́n Carlos
Published: October 2019

T-cell negative selection, a process by which intrathymic immunological tolerance is induced, involves the apoptosis-mediated clonal deletion of potentially autoreactive T cells. Although different experimental approaches suggest that this process is triggered as the result of activation-mediated cell death, the signal transduction pathways underlying this process is not fully understood. In the present report we have used an in vitro system to analyze the cell activation and proliferation requirements for the deletion of viral superantigen (SAg)-reactive Vβ8.1 T-cell receptor (TCR) transgenic (TG) thymocytes. Our results indicate that in vitro negative selection of viral SAg-reactive CD4+ CD8+thymocytes is dependent on thymocyte activation but does not require the proliferation of the negatively signaled thymocytes.
Treatment of Thrombocytopenia in Chimpanzees Infected With Human Immunodeficiency Virus by Pegylated Recombinant Human Megakaryocyte Growth and Development Factor
Authors: Harker Laurence A. , Marzec Ulla M. , Novembre Francis , Sundell I. Birgitta , Waller Edmund K. , Karpatkin Simon , McClure Harold M. , Kelly Andrew B. , Stead Richard B.
Published: October 2019

Three chimpanzees experimentally infected with human immunodeficiency virus (HIV) developed significant chronic thrombocytopenia after 5, 4, and 2 years, with peripheral platelet counts averaging 64 ± 19 × 103/μL (P = .004 compared with 228 ± 92 × 103/μL in 44 normal control animals), mean platelet volumes of 11.2 ± 1.8 fL (P > .5 compared with 10.9 ± 0.7 fL in normal controls), endogenous thrombopoietin (TPO) levels of 926 ± 364 pg/mL (P < .001 compared with 324 ± 256 pg/mL in normal controls), uniformly elevated platelet anti-glycoprotein (GP) IIIa49-66 antibodies, and corresponding viral loads of 534, 260, and 15 × 103 RNA viral copies/mL. Pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) was administered subcutaneously (25 μg/kg twice weekly for 3 doses) to determine the effects of stimulating platelet production on peripheral platelet concentrations in this cohort of thrombocytopenic HIV-infected chimpanzees. PEG-rHuMGDF therapy increased (1) peripheral platelet counts 10-fold (from 64 ± 19 to 599 ± 260 × 103 platelets/μL;P = .02); (2) marrow megakaryocyte numbers 30-fold (from 11.7 ± 6.5 × 106/kg to 353 ± 255 × 106/kg;P = .04); (3) marrow megakaryocyte progenitor cells fourfold (from a mean of 3.6 ± 0.6 to 14.1 × 103 CFU-Meg/1,000 CD34+ marrow cells); and (4) serum levels of Mpl ligand from 926 ± 364 pg/mL (endogenous TPO) to predosing trough levels of 1,840 ± 353 pg/mL PEG-rHuMGDF (P = .02). The peripheral neutrophil counts were also transiently increased from 5.2 ± 2.6 × 103/μL to 9.9 ± 5.0 × 103/μL (P= .01), but neither the erythrocyte counts nor the reticulocyte counts were altered significantly (P > .1). The serum levels of antiplatelet GPIIIa49-66 antibodies exhibited reciprocal reductions during periods of thrombocytosis (P < .07). PEG-rHuMGDF therapy did not increase viral loads significantly (395, 189, and 53 × 103 RNA viral copies/mL; P > .5 compared with baseline values). The striking increase in peripheral platelet counts produced by PEG-rHuMGDF therapy implies that thrombocytopenia in HIV-infected chimpanzees is attributable to insufficient compensatory expansion in platelet production resulting from HIV-impaired delivery of platelets despite stimulated megakaryocytopoiesis. These data suggest that PEG-rHuMGDF therapy may similarly correct peripheral platelet counts in thrombocytopenic HIV-infected patients.