bluuebunny/crossref_metadata_2025 huggingface dataset:
Gene transfer into human bone marrow hematopoietic cells mediated by adenovirus vectors [see comments]
Authors: Watanabe T , Kuszynski C , Ino K , Heimann DG , Shepard HM , Yasui Y , Maneval DC , Talmadge JE
Published: October 2019

Human bone marrow mononuclear cells (BMMNCs) and enriched CD34 positive (CD34+) cells were transduced with adenovirus vectors encoding Escherichia coli beta-galactosidase gene. Tranductions were carried out by 24-hour coincubation with adenovirus vectors at different multiplicities of infections (moi). Efficacy of gene transfer into BM cells and expression of the gene product (ie, beta-galactosidase) were studied using X-Gal histochemical staining and flow cytometric analysis. X-Gal staining demonstrated that the percentage of positive cells at mois of 5 to 500 was 3.4% to 34.5% for BMMNCs and 6.0% to 20.0% for enriched CD34+ cells. Similar results (1.5% to 35.7% for BMMNCs and 5.4% to 24.2% for enriched CD34+ cells) were obtained with flow cytometric analysis using fluorescein di-beta-D-galactopyranoside (FDG). Multicolor flow cytometry analysis, which included FDG, demonstrated that BM progenitors (CD34+ or CD34+CD38-), T cells (CD2+), B cells (CD19+), natural killer cells (CD56+), granulocytes, and monocytes all expressed the adenovirus transgene. To ascertain the effects of adenovirus vectors on normal BM progenitors, the numbers of colony forming unit-granulocyte/macrophage (CFU-GM), burst-forming unit- erythrocyte (BFU-E), and high-proliferative potential-colony-forming cells (HPP-CFC) after 24-hour coincubation with adenovirus vectors were determined. When BMMNCs or enriched CD34+ cells were incubated with adenovirus vectors at mois of 5 and 50, no significant differences in the numbers of CFU-GM, BFU-E, and HPP-CFC were observed compared with the uninfected control cells. However, the numbers of CFU-GM were significantly (P < .01) decreased when BMMNCs or enriched CD34+ cells were incubated with adenovirus vectors at a moi of 500, compared with the uninfected control cells. The adenovirus infected cells, purified by cell sorting for FDG expression, were capable of growing in culture and gave rise to various colonies (ie, CFU-GM, BFU-E, and HPP-CFC). These data indicate that recombinant adenovirus vectors can be used to transfer genes to human BM hematopoietic cells with expression of the exogenous gene at a high transduction efficiency.
A Thr359Met mutation in factor VII of a patient with a hereditary deficiency causes defective secretion of the molecule
Authors: Arbini AA , Mannucci M , Bauer KA
Published: October 2019

We elucidated the genetic basis responsible for factor VII deficiency in an Italian woman with a severe bleeding diathesis. In the allele inherited from the patient's father, we identified a G to A mutation at nucleotide 6070 at the 5′ splice site of intron 4 and a G to A substitution at nucleotide 10976 resulting in the Arg353Gln polymorphism. The maternal allele demonstrated a C to T substitution at nucleotide 10994 resulting in Thr359Met. The mutation at nucleotide 6070 alters an invariant GT dinucleotide and disrupts normal mRNA processing. To investigate the mechanism by which Thr359Met reduces factor VIl levels, we expressed wild type factor VII cDNA (FVIIwt) and a mutant factor VII cDNA containing the base substitution resulting in Met359 (FVII359M) in Chinese hamster ovary cells (CHO). In cells transfected with the mutant factor VII cDNA, FVII359M accumulated intracellularly, and no factor VII was detected in the media after 3 hours of chase. The carbohydrate side chains associated with FVII359M were sensitive to Endo H digestion, which indicates that the protein is retained in the endoplasmic reticulum. Analysis of cell lysates also showed that FVII359M was associated with the 78 kD protein corresponding to GRP78/BiP. We conclude that a Thr359Met mutation in factor VII results in a severe secretion defect that probably results from abnormal folding of the molecule.
Release of interleukin-12 in experimental Escherichia coli septic shock in baboons: relation to plasma levels of interleukin-10 and interferon- gamma
Authors: Jansen PM , van der Pouw Kraan TC , de Jong IW , van Mierlo G , Wijdenes J , Chang AA , Aarden LA , Taylor FB Jr. , Hack CE
Published: October 2019

Interleukin (IL)-12 is thought to be a key factor for the induction of interferon gamma (IFN-gamma), a cytokine essential for the lethal effects of endotoxin. We report here on the release of the nonfunctional subunit of IL-12, p40, as well as biologically active heterodimeric IL-12, p70, after administration of a lethal (n = 5) or sublethal (n = 8) dose of live Escherichia coli to baboons. Remarkably, on lethal challenge, peak levels of p40 were observed at 3 hours that were about twofold lower than those elicited after sublethal challenge (2,813 +/- 515 pg/mL v 4,972 +/- 732 pg/mL, P < .05). This disparity was also observed, although to a lesser extent, for IL-12 p70 antigen, of which maximum levels of 91 +/- 47 pg/mL and 151 +/- 41 pg/mL were measured 6 hours after a lethal or sublethal dose of E coli, respectively. Circulating p70 antigen correlated with IL-12 biologic activity (r = 0.869; P < .001). When comparing lethal to sublethal conditions, lower peak levels of IL-12 on lethal E coli sharply contrasted with higher levels of other proinflammatory cytokines, such as tumor necrosis factor (TNF)-alpha, IL-1beta, IL-6, and IL-8 observed in these animals. Lower IL-12 concentrations in the lethal group may have resulted in part from the enhanced production of IL-10, a known inhibitor of IL-12 synthesis in vitro, as peak levels of this cytokine 3 hours postchallenge inversely correlated with peak levels of IL-12, in particular p40 (r = -0.802; P < .01). Contrary to what might be expected if IFN-gamma were solely induced by IL-12, lethally challenged baboons generated threefold more IFN-gamma at 6 hours than those receiving a sublethal dose (P < .05). Moreover, higher levels of IFN- gamma were associated with lower p40/p70 ratios, suggesting that, in agreement with observations in vitro, IFN-gamma may have preferentially upregulated the release of p70 over p40. These data show that IL-12 is released in experimental septic shock in nonhuman primates and suggest that IL-10 and IFN-gamma are involved in the regulation of this release. Furthermore, this study indicates that the systemic release of IL-12 might be essential, but is not likely sufficient, to promote lethal production of IFN-gamma in sepsis.
p190 BCR-ABL mRNA is expressed at low levels in p210-positive chronic myeloid and acute lymphoblastic leukemias
Authors: van Rhee F , Hochhaus A , Lin F , Melo JV , Goldman JM , Cross NC
Published: October 2019

One hundred and forty-three patients with p210 BCR-ABL-positive leukemia were studied for coexpression of p190 BCR-ABL mRNA. p190 mRNA was detected in 14 of 16 (88%) patients with chronic-phase chronic myeloid leukemia (CML) at diagnosis, in 10 of 10 (100%) CML patients in blast crisis, in 75 of 107 (70%) CML patients receiving interferon- alpha (IFN-alpha), and 10 of 10 (100%) patients with p210 BCR-ABL- positive acute lymphoblastic leukemia (ALL). Neither p210 nor p190 BCR- ABL transcripts were detected in normal healthy adults (n = 20). The numbers of p190 transcripts determined by competitive PCR in patients with CML were low compared with the numbers of p210 transcripts. The median numbers of p210 and p190 transcripts per unit volume of cDNA in positive samples were 1.0 x 10(5) (range, 15 to 1.4 x 10(6)) and 10 (range, 10 to 2.9 x 10(3)), respectively. The numbers of p190 and p210 transcripts were significantly correlated in individual samples (r = .65, P < .001). The median number of p210 BCR-ABL transcripts was significantly lower in samples negative for p190 BCR-ABL transcripts than in samples in which p190 BCR-ABL transcripts were identified (3.1 x 10(3)[n = 73] v 1.0 x 10(5)[n = 115]; P < .0001). The median ratio of p190 to p210 BCR-ABL mRNA was not significantly different between chronic phase CML (1.9 x 10(-4)) and CML in blast crisis (1.7 x 10(- 4)). The median ratio in p210 ALL was also low (1.9 x 10(-3)) but significantly higher than that of CML. We conclude that pl90 BCR-ABL transcripts are frequently present at a low level in p210 BCR-ABL- positive leukemias. p190 mRNA may arise through alternative or missplicing and its presence is probably of no pathogenetic significance.
Macrophages can recognize and kill tumor cells bearing the membrane isoform of macrophage colony-stimulating factor
Authors: Jadus MR , Irwin MC , Irwin MR , Horansky RD , Sekhon S , Pepper KA , Kohn DB , Wepsic HT
Published: October 2019

NBXFO hybridoma cells produced both the membrane and secreted isoforms of macrophage colony-stimulating factor (M-CSF). Murine bone marrow cells stimulated by the secreted form of M-CSF (sM-CSF) became Mac1+, Mac2+, Mac3+, and F4/80+ macrophages that inhibited the growth of NBXFO cells, but not L1210 or P815 tumor cells. In cytotoxicity studies, M- CSF activated macrophages and freshly isolated macrophages killed NBXFO cells in the presence of polymyxin B, eliminating the possibility that contaminating lipopolysaccharide (LPS) was responsible for the delivery of the cytotoxic signal. Retroviral-mediated transfection of T9 glioma cells with the gene for the membrane isoform of M-CSF (mM-CSF), but not for the secreted isoform of M-CSF, transferred the ability of macrophages to kill these transfected T9 cells in a mM-CSF dose- dependent manner. Macrophage-mediated killing of the mM-CSF transfected clone was blocked by using a 100-fold excess of recombinant M-CSF. Catalase, superoxide dismutase, and the nitric oxide inhibitor, N-omega- nitro-arginine methyl ester (NAME), did not effect macrophage cytotoxicity against the mM-CSF transfectant T9 clones. T9 parental cells when cultured in the presence of an equal number of the mM-CSF transfectant cells were not killed, indicating specific target cell cytotoxicity by the macrophages. Electron microscopy showed that macrophages were capable of phagocytosizing mM-CSF bearing T9 tumor cells and NBXFO hybridoma cells; this suggested a possible mechanism of this cytotoxicity. This study indicates that mM-CSF provides the necessary binding and triggering molecules through which macrophages can initiate direct tumor cell cytotoxicity.
The stability of human beta-globin mRNA is dependent on structural determinants positioned within its 3' untranslated region
Authors: Russell JE , Liebhaber SA
Published: October 2019

Controls that act at both transcriptional and posttranscriptional levels assure that globin genes are highly expressed in developing erythroid cells. The extraordinary stabilities of alpha- and beta- globin mRNAs permit globin proteins to accumulate to substantial levels in these cells, even in the face of physiologic transcriptional silencing. Structural features that determine alpha-globin mRNA stability have recently been identified within its 3′UTR; in contrast, the structural features that determine beta-globin mRNA stability remain obscure. The current study begins to define the structural basis for beta-globin mRNA stability. Two tandem antitermination mutations are introduced into the wild-type human beta-globin gene that permit ribosomes to read into the 3′UTR of the encoded beta-globin mRNA. The readthrough beta-globin mRNA is destabilized in cultured erythroid cells, indicating that, as in human alpha-globin mRNA, an unperturbed 3′UTR is crucial to maintaining mRNA stability. Additional experiments show that the beta-globin and alpha-globin mRNA 3′UTRs provide equivalent levels of stability to a linked beta-globin mRNA coding region, suggesting a parallel in their functions. However, destabilization of the antiterminated beta-globin mRNA is independent of active translation into the 3′UTR, whereas translation into the alpha-globin mRNA 3′UTR destabilizes a linked beta-globin coding region in a translationally dependent manner. This indicates that the alpha- and beta-globin 3′UTRs may stabilize linked mRNAs through distinct mechanisms. Finally, it is shown that neither of the two mutations that, in combination, destabilize the beta-globin mRNA have any effect on beta-globin mRNA stability when present singly, suggesting potential redundancy of stabilizing elements. In sum, the current study shows that a functionally intact beta-globin mRNA 3′UTR is crucial to maintaining beta-globin mRNA stability and provides a level of stability that is functionally equivalent to, although potentially mechanistically distinct from, the previously characterized alpha- globin mRNA 3′UTR stability element.
Differential use of protein 4.1 translation initiation sites during erythropoiesis: implications for a mutation-induced stage-specific deficiency of protein 4.1 during erythroid development
Authors: Chasis JA , Coulombel L , McGee S , Lee G , Tchernia G , Conboy J , Mohandas N
Published: October 2019

Expression of multiple protein 4.1 isoforms in erythroid progenitors and in a variety of nonerythroid tissues results from alternative pre- mRNA splicing. In 4.1 pre-mRNA, several translation initiation sites are present; synthesis of isoforms larger than 80 kD occurs when an upstream 5′ AUG is spliced in, whereas the 80-kD mature erythroid isoform is produced when the upstream AUG is spliced out and translation is initiated at the downstream AUG. During erythropoiesis, this splicing switch is developmentally regulated. We studied this developmental switch in hereditary elliptocytosis 4.1Alg, in which a DNA rearrangement involving the exon containing the downstream AUG results in loss of coding capacity for the 80-kD 4.1, leading to mature red blood cells deficient in 4.1 with decreased membrane mechanical stability. Analysis of erythroblast RNA by reverse transcriptase- polymerase chain reaction showed that, although it retained the upstream AUG, its coding region was approximately 2.2 kb, compared with approximately 2.5 kb of normal 4.1 mRNA, because of the deletion of exons, including the one that codes for the downstream AUG. Immunofluorescent microscopy and Western blot analysis documented protein 4.1 expression in HE 4.1Alg erythroblasts. These studies emphasize the crucial role of differentiation-regulated RNA splicing because, within the same erythroid tissue, the HE 4.1Alg phenotype did not appear until after the differentiation-associated splicing event.
Hematopoietic defects in mice lacking the sialomucin CD34
Authors: Cheng J , Baumhueter S , Cacalano G , Carver-Moore K , Thibodeaux H , Thomas R , Broxmeyer HE , Cooper S , Hague N , Moore M , Lasky LA
Published: October 2019

Although the pluripotent hematopoietic stem cell can only be definitively identified by its ability to reconstitute the various mature blood lineages, a diversity of cell surface antigens have also been specifically recognized on this subset of hematopoietic progenitors. One such stem cell-associated antigen is the sialomucin CD34, a highly O-glycosylated cell surface glycoprotein that has also been shown to be expressed on all vascular endothelial cells throughout murine embryogenesis as well as in the adult. The functional significance of CD34 expression on hematopoietic progenitor cells and developing blood vessels is unknown. To analyze the involvement of CD34 in hematopoiesis, we have produced both embryonic stem (ES) cells and mice that are null for the expression of this mucin. Analysis of yolk saclike hematopoietic development in embryoid bodies derived from CD34- null ES cells showed a significant delay in both erythroid and myeloid differentiation that could be reversed by transfection of the mutant ES cells with CD34 constructs expressing either a complete or truncated cytoplasmic domain. Measurements of colony-forming activity of hematopoietic progenitor cells derived from yolk sacs or fetal livers isolated from CD34-null embryos also showed a decreased number of these precursor cells. In spite of these diminished embryonic hematopoietic progenitor numbers, the CD34-null mice developed normally, and the hematopoietic profile of adult blood appeared typical. However, the colony-forming activity of hematopoietic progenitors derived from both bone marrow and spleen is significantly reduced in adult CD34-deficient animals, and these CD34-deficient progenitors also appear to be unable to expand in liquid cultures in response to hematopoietic growth factors. Even with these apparent progenitor cell deficiencies, CD34- null animals showed kinetics of erythroid, myeloid, and platelet recovery after sublethal irradiation that are indistinguishable from wild-type mice. These data strongly suggest that CD34 plays an important role in the formation of progenitor cells during both embryonic and adult hematopoiesis. However, the hematopoietic sites of adult CD34-deficient mice may still have a significant reservoir of progenitor cells that allows for normal recovery after nonmyeloablative peripheral cell depletion.
High-frequency cell surface expression of a foreign protein in murine hematopoietic stem cells using a new retroviral vector
Authors: Tumas DB , Spangrude GJ , Brooks DM , Williams CD , Chesebro B
Published: October 2019

A retroviral vector (pSFF) derived from murine Friend spleen focus forming virus was used to transduce murine hematopoietic stem cells and express a cell surface marker protein, mutated murine prion protein, in vitro and in vivo after transplantation. To enhance retroviral vector integration in bone marrow cells, mice were treated with 5-fluorouracil (5-FU) to increase stem cell mitotic activity, which peaked on day 8 post-5-FU. The infectivity titer of the vector, pSFF-mPrP-3F4, was determined by a novel assay in which antigen-positive foci of infected cells were detected after replication and spread of the vector in cultures of mixed packaging cell lines. Infection of Sca-1+/Lineageneg- low cells with pSFF-mPrP-3F4 resulted in marker protein expression in 40% of the progeny cells after 7 days of culture. Transplantation of marrow cells or sorted Sca-1+/Lineageneg-low cells transduced with vector resulted in 3F4-positive mPrP expression in 11% to 37% of donor- derived peripheral blood leukocytes at 2 weeks. Though the percentage of 3F4-positive blood cells gradually declined, at 28 weeks 23% of recipient mice still maintained expression of the marker gene. Expression was observed in lymphoid, myeloid, and erythroid lineages and was detected in Sca-1+/Lineageneg-low marrow cells. The multilineage, high-frequency expression observed suggests that pSFF may be useful in gene therapy directed at hematopoietic stem cells and their differentiated progeny.
Human dendritic cell differentiation pathway from CD34+ hematopoietic precursor cells
Authors: Rosenzwajg M , Canque B , Gluckman JC
Published: October 2019

The most effective antigen-presenting cells for T lymphocytes are dendritic cells (DCs), the differentiation pathway of which, however, is incompletely characterized. We examined here how DCs differentiated from human cord blood CD34+ progenitor cells cultured with granulocyte- macrophage colony-stimulating factor, tumor necrosis factor-alpha, and stem cell factor. After 5 days, 2 of 3 nonadherent cells were CD13hiHLA- DRhiCD4+, half of them were also CD14+, and < or = 10% were CD1a+. When day-5 sorted CD13hiCD1a- and CD13lo cells were further cultured, CD1a+ cells appeared in the already CD13hi population, whereas CD13hi cells, a minority of which rapidly became CD1a+, emerged from the CD13lo population. By day 12, still 66% of bulk cells in suspension were CD13hi, most of which displayed high forward and side scatters of large granular cells. Half of CD13hi cells were CD1a+. All CD13hi cells expressed to the same extent DR, CD4, costimulatory and adhesion molecules, and various amounts of CD14. CD1a+ cells stimulated allogeneic lymphocytes more than CD13hiCD1a- cells and, although they were CD14+, both cell types were nonspecific esterase-negative nonphagocytic cells and were stronger mixed leukocyte reaction stimulators than were their macrophage counterparts. Eventually, the percentage of CD1a+ cells decreased. However, typical CD1a+ DCs still emerged in culture of sorted day-12 CD13hiCD1a- cells, and adding interleukin-4 to bulk cultures at that time led to the persistence of the CD1a+ population while diminishing CD14 expression. Thus, this system results first in the differentiation of CD13hi precursors that strongly express DR and CD4, from which more mature CD1a+ DCs continuously differentiate all along the culture period.
Thrombopoietin in thrombocytopenic mice: evidence against regulation at the mRNA level and for a direct regulatory role of platelets
Authors: Stoffel R , Wiestner A , Skoda RC
Published: October 2019

Thrombopoietin (TPO), originally described as an activity in the serum of thrombocytopenic animals that leads to increased production of platelets, has recently been isolated and cloned. Its closest relative in the cytokine superfamily, erythropoietin (EPO), is transcriptionally regulated during anemia, and it was expected that TPO would similarly be regulated during thrombocytopenia. We induced thrombocytopenia in mice and confirmed that TPO activity was upregulated, as determined by a bioassay. Liver and kidney were found to be the major sources of TPO mRNA. Surprisingly, TPO mRNA in these tissues was not upregulated in thrombocytopenic mice. Using a sensitive RNase protection assay that can distinguish between TPO isoforms, we found no change in the profile of mRNA for these isoforms. A semiquantitative reverse transcription- polymerase chain reaction assay also did not demonstrate upregulation of TPO mRNA in the spleen. Thus, the increase of TPO activity during thrombocytopenia is not caused by regulation at the level of TPO mRNA. Furthermore, isolated mouse platelets absorbed high amounts of bioactive TPO out of TPO-conditioned medium in a dose-dependent fashion. Our results are consistent with TPO protein being regulated at a posttranscriptional level and/or directly through absorption and metabolism by platelets.
Thrombin-induced GPIb-IX centralization on the platelet surface requires actin assembly and myosin II activation
Authors: Kovacsovics TJ , Hartwig JH
Published: October 2019

In resting platelets, the GPIb-IX complex, the receptor for the von Willebrand factor (vWF), is linked to underlying actin filaments by actin-binding protein (ABP-280). Thrombin stimulation of human platelets leads to a decrease in the surface expression of the GPIb-IX complex, which is redistributed from the platelet surface into the open canalicular system (OCS). Because the centralization of GPIb-IX is inhibited by cytochalasin, it is believed to be linked to actin cytoskeletal rearrangements that take place during platelet activation. We have further characterized the mechanism of GPIb-IX centralization in platelets in suspension. Following thrombin stimulation, GPIb-IX shifts from the membrane skeleton of the resting cell to the cytoskeleton of the activated cell in a reaction sensitive to cytochalasin B. The cytoskeletal association of GPIb-IX involves ABP- 280, as it correlates with the incorporation of ABP-280 into the activated cytoskeleton and because no dissociation of the ABP-280/GPIb- IX complexes is detected after thrombin activation. However, the incorporation of GPIb-IX into the cytoskeleton is complete within 1 minute, whereas GPIb-IX centralization requires 5 to 10 minutes for completion. The movement of GPIb-IX to the cytoskeleton of activated platelets is therefore necessary, but not sufficient for GPIb-IX centralization. Blockage of cytosolic calcium increases induced by thrombin by loading with the cell permeant calcium chelator Quin-2 AM inhibited GPIb-IX centralization by 70%, but did not prevent its association with the activated cytoskeleton. Quin-2 loading did, however, decrease the incorporation of myosin II into the activated cytoskeleton. The role of myosin II was further probed using the myosin light chain kinase (MLCK) inhibitor wortmannin. Wortmannin prevents myosin II association to the activated cytoskeleton and inhibits GPIb- IX centralization by 50%, without affecting actin assembly or the association of GPIb-IX to the cytoskeleton. Only micromolar concentrations of wortmannin, high enough to inhibit MLCK, prevent GPIb- IX centralization. These results indicate that thrombin-induced GPIb-IX centralization requires a minimum of two steps, one associating GPIb-IX to the activated cytoskeleton and the second requiring myosin II activation. The involvement of myosin II implies that GPIb-IX/ABP-280 complexes, linked to actin filaments, are pulled into the cell center, and that platelets may exert contractile tension on vWF bound to its receptor.
Molecular cloning and expression of murine vascular endothelial- cadherin in early stage development of cardiovascular system
Authors: Breier G , Breviario F , Caveda L , Berthier R , Schnurch H , Gotsch U , Vestweber D , Risau W , Dejana E
Published: October 2019

An early step in the formation of the extraembryonic and intraembryonic vasculature is endothelial cell differentiation and organization in blood islands and vascular structures. This involves the expression and function of specific adhesive molecules at cell-to-cell junctions. Previous work showed that endothelial cells express a cell-specific cadherin (vascular endothelial [VE]-cadherin, or 7B4/cadherin-5) that is organized at cell-to-cell contacts in cultured cells and is able to promote intercellular adhesion. In this study, we investigated whether VE-cadherin could be involved in early cardiovascular development in the mouse embryo. We first cloned and sequenced the mouse VE-cadherin cDNA. At the protein level, murine VE-cadherin presented 75% identity (90%, considering conservative amino acid substitutions) with the human homologue. Transfection of murine VE-cadherin cDNA in L cells induced Ca(++)-dependent cell-to-cell aggregation and reduced cell detachment from monolayers. In situ hybridization of adult tissues showed that the murine molecule is specifically expressed by endothelial cells. In mouse embryos, VE-cadherin transcripts were detected at the very earliest stages of vascular development (E7.5) in mesodermal cells of the yolk sac mesenchyme. At E9.5, expression of VE-cadherin was restricted to the peripheral cell layer of blood islands that gives rise to endothelial cells. Hematopoietic cells in the center of blood islands were not labeled. At later embryonic stages, VE-cadherin transcripts were detected in vascular structures of all organs examined, eg, in the ventricle of the heart, the inner cell lining of the atrium and the dorsal aorta, in intersomitic vessels, and in the capillaries of the developing brain. A comparison with flk-1 expression during brain angiogenesis revealed that brain capillaries expressed relatively low amounts of VE-cadherin. In the adult brain, the level of VE-cadherin transcript was further reduced. By immunohistochemistry, murine VE-cadherin protein was detected at cell-to-cell junctions of endothelial cells. Overall, these data demonstrate that VE-cadherin is an early, constitutive, and specific marker of endothelial cells. This distinguishes this molecule from other cadherins and suggests that its expression is associated with the early assembly of vascular structures.
The ontogeny of key endoplasmic reticulum proteins in human embryonic and fetal red blood cells
Authors: Hume R , Burchell A , Allan BB , Wolf CR , Kelly RW , Hallas A , Burchell B
Published: October 2019

Recently, using immunohistochemical methods, we surprisingly found that endoplasmic reticulum glucose-6-phosphatase is present in human embryonic and fetal red blood cells (RBCs) but not in adult RBCs. The fact that an endoplasmic reticulum enzyme, whose major site of expression in adults is the liver, is present in human embryonic and fetal RBCs, particularly nucleated cells, indicated that it would be sensible to determine whether these cells also contain other endoplasmic reticulum enzyme systems normally found in adult liver. Therefore, we have studied the expression of other endoplasmic reticulum proteins and found that human embryonic and fetal RBC precursors contain other protein components of the glucose-6- phosphatase system, ie, the phosphate and glucose transport proteins as well as other enzymes (eg, uridine diphosphate- glucuronosyltransferases, cytochrome P450 isozymes, nicotinamide adenine dinucleotide phosphate cytochrome P450 oxidoreductase, and prostaglandin H synthase). In addition, we also found the predominantly cytosolic markers 15-hydroxyprostaglandin dehydrogenase, prostaglandins PGE2 and 13,14-dihydro-15-keto-PGE2. The expression of key enzymes that control glucose production, detoxification of endobiotics and xenobiotics, and the regulation of prostaglandin levels in embryonic and early fetal RBCs means that these cells may have an important role in protecting the developing conceptus before it establishes an efficient circulation and before all tissues fully express their normal complement of these enzymes.
The probability of finding a suitable related donor for bone marrow transplantation in extended families [see comments]
Authors: Schipper RF , D'Amaro J , Oudshoorn M
Published: October 2019

In bone marrow transplantation, the advantages of family donors over unrelated donors are threefold. (1) Family donors are better matched because they share complete haplotypes. (2) The time between the start of the search and the actual transplantation can be much shorter than for unrelated donors. (3) Related bone marrow transplantation is cheaper. We developed a procedure for calculating the probability of finding a suitable donor among cousins and blood-related aunts and uncles (the extended family). The procedure calculates the following probabilities (P): (1) P that any blood-related uncle or aunt is a suitable donor, (2) P that a suitable donor exists in all blood-related uncles/aunts (from [1]), (3) P that any cousin is a suitable donor (as in [1]), (4) P that a suitable donor exists in all cousins (from [3]), (5) P that a suitable donor exists in the entire extended family (from [2] and [4]). Additionally, we discuss the suitability of this procedure in the daily practice of donor procurement. The procedure is also suitable to search for family donors who have a single antigen mismatch with the patient. We also discuss the differences between our method and the one recently described by Kaufman (Bone Marrow Transplant 15:279, 1995).
The origin of IgG production and homogeneous IgG components after allogeneic bone marrow transplantation
Authors: van Tol MJ , Gerritsen EJ , de Lange GG , van Leeuwen AM , Jol-van der Zijde CM , Oudeman-Gruber NJ , de Vries E , Radl J , Vossen JM
Published: October 2019

Pediatric recipients (n = 25) of an allogeneic bone marrow (BM) graft were selected on the basis of informative IgG allotype (Gm) differences between the BM donor and the recipient. To investigate the kinetics of the appearance of IgG of donor origin and the disappearance of IgG of recipient origin, G1m and G2m allotype levels were quantified in sera obtained at regular intervals between 3 months and 5 years after BM transplantation (BMT). For this quantification, a dot immunobinding assay (DIBA) has been developed. In 19 of 22 informative recipients, the Gm allotype distribution had reached the range of values expected on the basis of the Gm phenotype of the donor within 6 months after BMT. Remarkably, IgG of recipient origin persisted in 15 of 18 informative recipients until last follow up, ie, for several years after BMT. In addition to the origin of total IgG production, the origin of homogeneous IgG components (H-IgG) appearing after BMT was investigated. H-IgG of donor origin could be detected as early as 3 weeks after BMT, but also H-IgG of recipient origin were present in 8 of 13 informative recipients for a period of up to 1 year after BMT. We conclude that host-type IgG-producing cells were not eradicated by the (myeloablative) conditioning regimen and persisted in a high number of graft recipients. It is our hypothesis that lack of graft-versus-host disease (GVHD) in the majority of these recipients results in the persistence of IgG-producing cells of host origin. These observations may be relevant for the evaluation of patients who received allogeneic BMT for the treatment of multiple myeloma.
High incidence of potential p53 inactivation in poor outcome childhood acute lymphoblastic leukemia at diagnosis
Authors: Marks DI , Kurz BW , Link MP , Ng E , Shuster JJ , Lauer SJ , Brodsky I , Haines DS
Published: October 2019

Previous studies have indicated that p53 gene mutations were an uncommon event in acute lymphoblastic leukemia (ALL) in children. In one series of 330 patients, p53 mutations were seen in fewer than 3%. We analyzed bone marrow mononuclear cells derived from 10 children with ALL at diagnosis who subsequently failed to achieve a complete remission or who developed relapse within 6 months of attaining complete remission for p53 gene mutations and mdm-2 overexpression. We found that three children had p53 gene mutations, and four overexpressed mdm-2. Also, experiments comparing relative levels of mdm- 2 RNA and protein in these patients demonstrated that mdm-2 overexpression can occur at the transcriptional and posttranscriptional level in primary leukemic cells. Although we were unable to link Waf-1 RNA expression with p53 status in childhood ALL, our data show potential p53 inactivation by multiple mechanisms in a large percentage of these patients and demonstrate that these alterations can be detected at diagnosis. Inactivation of the p53 pathway may, therefore, be important in children with ALL who fail to respond to treatment and may be useful for the early identification of children requiring alternative therapies.
Leukemia-associated changes identified by quantitative flow cytometry. IV. CD34 overexpression in acute myelogenous leukemia M2 with t(8;21)
Authors: Porwit-MacDonald A , Janossy G , Ivory K , Swirsky D , Peters R , Wheatley K , Walker H , Turker A , Goldstone AH , Burnett A
Published: October 2019

During the immunodiagnosis of 517 cases of acute myelogenous leukemia (AML) entered into the Medical Research Council (MRC) AML 10 trials, we have observed the CD34 precursor cell antigen more frequently in AML of M2 morphology, especially in the 84% of cases with the t(8;21) chromosomal translocation, than in any other French-American-British classification group. CD34 expression was then quantified (using QIFI and Quantum Simply Cellular beads [Flow Cytometry Standards, Research Triangle Park, NC] and CD34+ standard cells). When CD34 antibody- binding capacity (ABC) of normal bone marrow (BM) precursors and leukemic blasts was compared, it was shown that AML M2 cases with t(8;21) not only had the highest percentages of CD34+ blasts, but in > 80% of CD34+ cases the individual blasts expressed higher than normal levels of CD34 antigen (> 60 x 10(3) ABC per cell). In addition, in 73% of this group CD34 antigen was overexpressed in an asynchronous combination with cytoplasmic myeloperoxidase (MPO). Other signs of asynchrony included high CD34 expression with CD15 and/or CD56, as well as aberrant combinations of CD13 with terminal deoxynucleotidyl transferase (TdT) and CD19. These findings demonstrate that asynchrony is identifiable in virtually every case of AML with t(8;21), although it does not always involve the same antigens. M2 cases with t(8;21), mostly CD34+, had a 100% remission rate and 71% 5-year survival rate; other patients with CD34+ or CD34- AML showed 69% and 84% remission rates and 31% and 36% 5-year survival rates, respectively. Consequently, individual markers such as CD34 should be interpreted in relation to other features such as chromosomal changes. These simple methods, which are well suited to quantify the expression of ligands, are a useful contribution to diagnosis: 60% to 65% of M2 cases with t(8;21) are rapidly identified by CD34 overexpression alone. This aberration, together with the other signs of asynchrony seen at presentation, can be used to search for residual leukemia after therapy.
Detection of altered membrane phospholipid asymmetry in subpopulations of human red blood cells using fluorescently labeled annexin V
Authors: Kuypers FA , Lewis RA , Hua M , Schott MA , Discher D , Ernst JD , Lubin BH
Published: October 2019

The phospholipids of the human red cell are distributed asymmetrically in the bilayer of the red cell membrane. In certain pathologic states, such as sickle cell anemia, phospholipid asymmetry is altered. Although several methods can be used to measure phospholipid organization, small organizational changes have been very difficult to assess. Moreover, these methods fail to identify subpopulations of cells that have lost their normal phospholipid asymmetry. Using fluorescently labeled annexin V in flow cytometry and fluorescent microscopy, we were able to identify and quantify red cells that had lost their phospholipid asymmetry in populations as small as 1 million cells. Moreover, loss of phospholipid organization in subpopulations as small as 0.1% of the total population could be identified, and individual cells could be studied by fluorescent microscopy. An excellent correlation was found between fluorescence-activated cell sorter (FACS) analysis results using annexin V to detect red cells with phosphatidylserine (PS) on their surface and a PS-requiring prothrombinase assay using similar red cells. Cells that bound fluorescein isothiocyanate (FITC)-labeled annexin V could be isolated from the population using magnetic beads covered with an anti-FITC antibody. Evaluation of blood samples from patients with sickle cell anemia under oxygenated conditions demonstrated the presence of subpopulations of cells that had lost phospholipid asymmetry. While only a few red cells were labeled in normal control samples (0.21% +/- 0.12%, n = 8), significantly increased (P < .001) annexin V labeling was observed in samples from patients with sickle cell anemia (2.18% +/- 1.21%, n = 13). We conclude that loss of phospholipid asymmetry may occur in small subpopulations of red cells and that fluorescently labeled annexin V can be used to quantify and isolate these cells.
Effects of granulocyte colony-stimulating factor on plasma cytokine and cytokine receptor levels and on the in vivo host response to endotoxin in healthy men
Authors: Pollmacher T , Korth C , Mullington J , Schreiber W , Sauer J , Vedder H , Galanos C , Holsboer F
Published: October 2019

We investigated the effects of granulocyte colony-stimulating factor (G- CSF) on cytokine and cytokine receptor plasma levels and on the in vivo host response to Salmonella abortus equi endotoxin in healthy males. Twenty volunteers received 0.8 ng/kg endotoxin and saline intravenously 1 week apart in randomized order. Twelve hours before both experiments, 10 of these subjects were pretreated with 300 micrograms G-CSF subcutaneously. G-CSF itself increased granulocyte and monocyte counts and the plasma levels of tumor necrosis factor-alpha (TNF-alpha), soluble TNF receptors (sTNF-R) p55, and p75 and interleukin-1 receptor antagonist (IL-1ra). G-CSF did not influence plasma IL-1 beta and IL-6 levels. In the G-CSF-pretreated subjects endotoxin-induced surges in rectal temperature and in the plasma levels of TNF-alpha plasma levels were about 50% increased, and surges in the plasma levels of both sTNF- Rs and IL-1ra were about twice as high as in the control group. Endotoxin-induced increases in IL-6, cortisol, and heart rate were not modified by G-CSF pretreatment. Endotoxin administration induced a transient 50% reduction in leukocyte counts in the G-CSF-pretreated subjects that was not seen in the control group. We conclude that a single stand dose of G-CSF increases the plasma levels of cytokines and cytokine receptors and considerably modifies the host response of healthy humans to a low dose of endotoxin.
Fanconi anemia genes act to suppress a cross-linker-inducible p53- independent apoptosis pathway in lymphoblastoid cell lines
Authors: Kruyt FA , Dijkmans LM , van den Berg TK , Joenje H
Published: October 2019

Hypersensitivity to cross-linking agents such as mitomycin C (MMC) is characteristic of cells from patients suffering from the inherited bone marrow failure syndrome. Fanconi anemia (FA). Here, we link MMC hypersensitivity of Epstein-Barr virus (EBV)-immortalized FA lymphoblasts to a high susceptibility for apoptosis and p53 activation. In MMC-treated FA cells belonging to complementation group C (FA-C), apoptosis followed cell cycle arrest in the G2 phase. In stably transfected FA-C cells, plasmid-driven expression of the wild-type cytoplasmic FAC protein relieved MMC-dependent G2 arrest and suppressed p53 activation. However, in both FA and non-FA lymphoblasts, p53 seemed not to be instrumental in the induction of MMC-dependent apoptosis, since overexpression of a dominant-negative p53 mutant failed to affect cell survival. In addition, no differences in the level of Bcl-2 expression, an inhibitor of apoptosis, were detected between FA and non- FA cells either in the absence or presence of MMC. Our findings suggest that FAC and the other putative FA gene products may function in a yet to be identified p53-independent apoptosis pathway.
The role of granzyme B in murine models of acute graft-versus-host disease and graft rejection
Authors: Graubert TA , Russell JH , Ley TJ
Published: October 2019

A complete molecular description of the syndromes of graft-versus-host disease (GVHD) and graft rejection could have a significant impact on clinical bone marrow transplantation. Recent in vitro experiments (Heusel et al, Cell 76:977, 1994 and Shresta et al, Proc Natl Acad Sci USA 92:5679, 1995) have shown that the putative mediators of these two syndromes, cytotoxic lymphocytes (CTL) and natural killer (NK) cells, respectively, initiate a program of cell death (apoptosis) in susceptible target tissues in a manner critically dependent on the serine protease Granzyme B (gzm B). In the present study, we have analyzed the phenotype of gzm B-deficient mice using experimental transplant models designed to isolate their CD8+ CTL, CD4+ CTL, and NK compartments. We found a significant impairment in class I-dependent GVHD mediated by gzm B -/- CD8+ CTL, whereas class II-dependent GVHD was not altered using gzm B -/- CD4+ effectors. In a hybrid resistance model, gzm B -/- hosts rejected haplo-identical marrow grafts as efficiently as did their wild-type littermates. This result is surprising in light of a severe defect in the ability of gzm B -/- NK cells to induce apoptosis in susceptible targets in vitro. These in vivo data define significant role for gzm B in cytotoxicity mediated by CD8+ CTL, but not by CD4+ CTL. Furthermore, these results do not support a model of hybrid resistance in which NK cells play a pivotal role.
Biosynthetic defect in platelet glycoprotein IX mutants associated with Bernard-Soulier syndrome
Authors: Sae-Tung G , Dong JF , Lopez JA
Published: October 2019

To evaluate the biosynthetic basis for decreased glycoprotein (GP) Ib- IX expression resulting from GP IX mutations described in three siblings with Bernard-Soulier syndrome, we introduced each mutation into the cDNA for GP IX by site-directed mutagenesis (GP IX Asp21 --> Gly and GP IX Asn45 --> Ser) and examined the associations of the mutants with the two other subunits of the GP Ib-IX complex in transfected cells. Unlike wild-type GP IX, neither of the mutants was able to increase GP Ib expression on the cell surface, either when transfected into Chinese hamster ovary (CHO) alpha beta cells or when cotransfected with GP Ib alpha and GP Ib beta into wild-type CHO cells. We also evaluated whether cotransfecting wild-type or mutant GP IX with GP Ib beta would result in the appearance of GP IX on the surface of the transfected cells; the wild-type protein was detected on the surface of the cells, whereas neither mutant reached the cell surface in appreciable quantities. Immunofluorescence microscopy of permeabilized cells revealed that the failure to express mutant GP IX on the cell surface did not result from failure to synthesize the polypeptide. Both mutants were detected in intracellular compartments, albeit at lower levels than the wild-type polypeptide (the fluorescence of cells expressing the GP IX Asp21 --> Gly was consistently the lowest). Direct evidence that the mutants associate poorly with Gp Ib beta was obtained of 35S-labeled cells transiently expressing GP Ib beta and wild-type or mutant GP IX. The amount of GP IX coprecipitated with GP Ib beta was greatly diminished in cells expressing either mutant. These findings suggest an important role for the conserved leucine-rich motif of GP IX in the association of this polypeptide with GP Ib beta and provide further evidence for the importance of GP IX in the stability of the GP-Ib-IX complex.
Human interferon-inducible protein-10 induces mononuclear cell infiltration in mice and promotes the migration of human T lymphocytes into the peripheral tissues and human peripheral blood lymphocytes-SCID mice
Authors: Taub DD , Longo DL , Murphy WJ
Published: October 2019

The human cytokine, interferon-inducible protein-10 (IP-10), is a small glycoprotein secreted by activated monocytes, T cells, keratinocytes, astrocytes, and endothelial cells and is structurally related to the alpha subfamily of chemotactic cytokines called chemokines (Taub and Oppenheim, Cytokine 5:175, 1993). However, in contrast to other alpha chemokines that induce neutrophil migration, IP-10 has been shown to chemoattract monocytes and T lymphocytes in vitro, suggesting a role in T-cell-mediated immune responses. We therefore examined the effects of human IP-10 after in vivo administration. IP-10 induces significant mononuclear cell infiltration after subcutaneous injections in normal mice. In an effort to study the in vivo effects of IP-10 on human leukocyte migration, we then examined the ability of recombinant human IP-10 (rhIP-10) to induce human-T-cell infiltration using a human/severe combined immune deficiency (SCID) mouse model. SCID mice received an intraperitoneal injection of human peripheral blood lymphocytes (10(8) cells), followed by a subcutaneous injection of rhIP- 10 (1 micrograms/injection) in the hind flank for 4 hours or sequential injections for 3 days. The skin and underlying tissue from the rhIP-10 injection site were then biopsied and examined for the extent of mononuclear cell infiltration. rhIP-10 again induced significant mononuclear cell accumulation 72 hours after injection. Immunohistologic evaluation determined that a significant number of human CD3+ T cells were recruited in response to rhIP-10 injections. These results show that rhIP-10 is capable of inducing human T-cell migration in vivo and may play an important role in monocyte and lymphocyte recruitment into inflammatory sites.