bluuebunny/crossref_metadata_2025 huggingface dataset:
Human Non-Hodgkin's Lymphomas Overexpress a Wild-Type Form of p53 Which Is a Functional Transcriptional Activator of the Cyclin-Dependent Kinase Inhibitor p21
Authors: Maestro Roberta , Gloghini Annunziata , Doglioni Claudio , Piccinin Sara , Vukosavljevic Tamara , Gasparotto Daniela , Carbone Antonino , Boiocchi Mauro
Published: October 2019

A large fraction of non-Hodgkin's lymphomas (NHLs) accumulate a wild-type form of the p53 tumor suppressor protein at the nuclear level. In normal cells, p53 induction is associated with a temporary cell growth arrest at the G1-S boundary of the cell cycle. This activity of p53 as a G1 checkpoint molecule is strictly dependent on its ability to induce the transcription of the inhibitor of the cyclin dependent kinase, p21. To verify the functionality of the wild-type p53 protein accumulated in NHL cells, 70 cases were comparatively analyzed for p53 and p21 expression and status of the respective genes. Overexpression of the wt p53 protein was associated with the accumulation of p21, indicating that p53 is functional with respect to p21 induction in these tumors. The coaccumulation of p53 with Ki-67 antigen indicates that wt p53-positive cells and p21-positive cells, as well, are actively proliferative elements, supporting the notion that p53-induced, p21-mediated growth arrest is somehow overridden in NHL cells. No p21 mutation or particular allele variant was shown to correlate with p21 protein accumulation, thus excluding a role for p21 structural abnormalities. Taken together, our data suggest the existence in NHL of a peculiar mechanism of functional inactivation of the p53 G1 checkpoint pathway occurring downstream of the CDK inhibitor p21.
Increased Circulating Colony-Stimulating Factor-1 (CSF-1) in SJL/J Mice With Radiation-Induced Acute Myeloid Leukemia (AML) Is Associated With Autocrine Regulation of AML Cells by CSF-1
Authors: Haran-Ghera Nechama , Krautghamer Rita , Lapidot Tsvee , Peled Alpha , Dominguez Melissa G. , Stanley E. Richard
Published: October 2019

The SJL/J mouse strain has a high spontaneous incidence of a B-cell neoplasm, reticulum cell neoplasm type B (RCN B). In addition, following irradiation, 10% to 30% of these mice develop acute myelomonocytic leukemia (radiation-induced acute myeloid leukemia [RI-AML]), an incidence that can be increased to 50% by treatment of the mice with corticosteroids after irradiation. The role played by the mononuclear phagocyte growth factor, colony-stimulating factor-1 (CSF-1), in the development of RI-AML in SJL/J mice was investigated. Mice dying of RI-AML, but not those dying of RCN B or without disease, possessed elevated concentrations of circulating CSF-1. In addition, in mice developing RI-AML with a more prolonged latency, circulating CSF-1 concentrations were increased before overt expression of RI-AML. First-passage tumors from 14 different RI-AMLs all contained high concentrations of CSF-1, and six of six different first- or second-passage tumors expressed the CSF-1 receptor (CSF-1R). Furthermore, in vitro colony formation by first- or second-passage tumor cells from 20 of 20 different RI-AMLs was blocked by neutralizing anti–CSF-1 antibody, and four of four of these tumors were inhibited by anti–CSF-1R antibody. The results of these antibody neutralization studies, coupled with the observation of elevated circulating CSF-1 in mice developing RI-AML, show an autocrine role for CSF-1 in RI-AML development in SJL/J mice. Southern blot analysis of tumor DNA from six of six of these tumors failed to reveal any rearrangements in the genes for CSF-1 or the CSF-1R. Studies in humans have shown that patients with AML possess elevated levels of circulating CSF-1 and that AML cells can express CSF-1 and the CSF-1R. Thus, RI-AML in the SJL/J mouse appears to be a useful model for human AML.
Inhibition of Sickle Erythrocyte Adhesion to Immobilized Thrombospondin by von Willebrand Factor Under Dynamic Flow Conditions
Authors: Barabino Gilda A. , Wise Robert J. , Woodbury Valerie A. , Zhang Beibing , Bridges Kenneth A. , Hebbel Robert P. , Lawler Jack , Ewenstein Bruce M.
Published: October 2019

Sickle red blood cell (RBC) adhesion to the blood vessel wall is hypothesized to be the initiating event in the periodic vaso-occlusive episodes that characterize sickle cell disease (SCD). Thrombospondin-1 (TSP) and von Willebrand factor (vWF ) have each been implicated in the adhesion of sickle RBC to vascular endothelial cells (EC) and subendothelial matrices. To better understand the contributions of each of these adhesive glycoproteins, we examined the adhesion of sickle RBC to immobilized TSP and vWF using a parallel plate flow chamber. Under postcapillary venular shear stress (1 dyne/cm2), sickle RBC adhered preferentially to TSP. To explore potential interactive effects of vWF and TSP, we examined sickle RBC adhesion to mixtures of these proteins. Whether the proteins were first combined in solution or sequentially applied to the slide, the presence of vWF inhibited the binding of sickle RBC to TSP. The inhibition of adhesion by vWF was shown to be the result of specific and saturable binding of vWF to TSP. Furthermore, vWF in solution at normal plasma levels also inhibited RBC adhesion to immobilized TSP. These data indicate that sickle RBC adhesion in vivo may be significantly influenced by the relative concentrations of TSP and vWF in the vascular wall.
Evidence of Genetic Diversity Underlying Rh D−, Weak D (Du), and Partial D Phenotypes as Determined by Multiplex Polymerase Chain Reaction Analysis of the RHD Gene
Authors: Avent Neil D. , Martin Peter G. , Armstrong-Fisher Sylvia S. , Liu Wendy , Finning Kirstin M. , Maddocks Deborah , Urbaniak Stanislaw J.
Published: October 2019

The human blood group Rh antigens are expressed by proteins encoded by a pair of highly homologous genes located at chromosome 1p34-36. One of the genes (RHCE ) encodes Rh CcEe antigens, while the other (RHD) the D antigen. Point mutations in the RHCE gene generate the C/c and E/e polymorphisms, while it has been shown that an RHD gene deletion can generate the D-negative phenotype. We have analyzed intron 4 of the RHCE and RHD genes and have defined the site of an RHD-specific deletion located in this intron. Using a multiplex RHD typing assay, which combines a reverse polymerase chain reaction (PCR) primer, which straddles this RHD-specific sequence, and a pair of primers located in exon 10 of the RHD gene, we have analyzed 357 different genomic DNA samples derived from individuals expressing D+, D−, weak D, and partial D phenotypes. Of these, we have noted a significant discordance with our multiplex PCR assay in the D− phenotypes dCcee and dccEe (which have been previously described) and weak D phenotypes. Our results suggest that in five serologically D− individuals we have identified an apparently intact RHD gene. Sequence analysis of transcripts obtained from one of these individuals (of phenotype dCCee) illustrates the presence of full-length RHD transcripts, which have a point mutation at nucleotide 121 (C → T), which generates an in-frame stop codon (Gln41Stop). Thus, we describe a different molecular basis for generating the D− phenotype to the complete RHD gene deletion described previously. We also show that there are discordances with serotype and the multiplex assay in weak D and partial D phenotypes, indicating that the underlying molecular basis can be heterogeneous. Existing Rh D PCR assays assume the complete absence of the RHD gene in D− phenotypes. We describe a different molecular basis for generating the D− phenotype to the complete RHD gene deletion described previously.
Stem Cell Transplantation for Secondary Acute Myeloid Leukemia: Evaluation of Transplantation as Initial Therapy or Following Induction Chemotherapy
Authors: Anderson Jeanne E. , Gooley Ted A. , Schoch Gary , Anasetti Claudio , Bensinger William I. , Clift Reginald A. , Hansen John A. , Sanders Jean E. , Storb Rainer , Appelbaum Frederick R.
Published: October 2019

The purpose of this report is to describe the results of stem cell transplantation as initial treatment for secondary acute myeloid leukemia (AML). Forty-six patients (median age 42 years) with secondary AML (17 therapy-related, 29 myelodysplasia-related) who had not received remission induction chemotherapy underwent allogeneic (n = 43) or syngeneic (n = 3) transplantation. The 5-year actuarial disease-free survival was 24.4%, and the cumulative incidences of relapse and nonrelapse mortality were 31.3% and 44.3%, respectively. Lower peripheral blood blast count was associated with a lower risk of relapse (P = .05) and shorter time from AML diagnosis to transplant was associated with a lower risk of nonrelapse mortality (P = .02) and improved disease-free survival (P = .026). Patients with therapy-related secondary AML tended to have lower disease-free survival (P = .16) and a higher relapse rate (P = .16) than patients whose leukemia was not therapy-related. The results of these 46 previously untreated patients were compared to 20 patients (median age 36 years, 12 therapy-related, 8 myelodysplasia-related) transplanted with chemotherapy-sensitive disease after induction chemotherapy (first complete remission [n = 6], second complete remission [n = 3], first untreated relapse [n = 11]). We found no statistically significant difference in outcome between these 2 groups of patients. These results suggest that prompt transplantation should be considered after diagnosis of secondary AML or, if possible, high-risk myelodysplasia, particularly in patients with low peripheral blast counts. Innovative transplant strategies are needed to reduce the high risks of relapse and nonrelapse mortality seen in this patient population.
Granulocyte Colony-Stimulating Factor–Induced Comobilization of CD4−CD8− T Cells and Hematopoietic Progenitor Cells (CD34+) in the Blood of Normal Donors
Authors: Kusnierz-Glaz Claus R. , Still Beverley J. , Amano Masahiko , Zukor Jazil D. , Negrin Robert S. , Blume Karl G. , Strober Samuel
Published: October 2019

The feasibility of transplantation of HLA-matched hematopoietic progenitor cells from the blood of normal donors given granulocyte colony-stimulating factor (G-CSF ) has been reported recently. In the current study, the changes in T-cell subsets as well as CD34+ cells were determined in one blood volume leukapheresis products of six normal individuals given G-CSF. Examination of the T-cell subsets in the leukapheresis products showed three different patterns: one in which a discrete population of CD4−CD8−αβ T cells was found in addition to the typical CD4+ and CD8+ T cells in the unfractionated as well as in high- and low-density cells; a second in which the discrete population of CD4−CD8−αβ T cells was predominant only in the low-density fractions; and a third in which a discrete population of CD4−CD8− T cells was not observed. The median yield of CD4−CD8− T cells was about fourfold to fivefold higher than the calculated number present in one blood volume (5L) from normal individuals. The ratios of CD34+ cells to CD4+ and CD8+ T cells, and of CD4−CD8− T cells to CD4+ and CD8+ T cells, were highest in the low-density fractions. These fractions suppressed the mixed leukocyte, and may ameliorate graft-versus-host disease as compared with unfractionated cells.
p53 Is Not Required for Regulation of Apoptosis or Radioprotection by Interleukin-3
Authors: Silva Augusto , Wyllie Andrew , Collins Mary K.L.
Published: October 2019

Primary interleukin-3 (IL-3)–dependent mast cell cultures from bone marrow of p53-null mice and littermate controls were established. Both p53-null and wild-type cells entered apoptosis on IL-3 removal, showing that p53 is not required for entry into apoptosis after factor deprivation. After X-irradiation, a lower proportion of the p53-null than wild-type cells underwent G2 arrest, but their radiosensitivity was similar. An IL-3–dependent cell line expressing wild-type p53 was used to show that cells die at a fixed time after X-irradiation rather than from a specific cell cycle point.
Mammalian Homeobox B6 Expression Can Be Correlated With Erythropoietin Production Sites and Erythropoiesis During Development, But Not With Hematopoietic or Nonhematopoietic Stem Cell Populations
Authors: Zimmermann Frank , Rich Ivan N.
Published: October 2019

There has been increasing interest in the involvement of mammalian homeobox (HOX) genes in hematopoietic regulation. The HOX genes are clustered in 4 chromosomes in mice and humans. In general, 5′ end HOX gene expression is predominant in hematopoietic stem cell populations, whereas 3′ end HOX gene expression are primarily found in committed progenitor cells. Furthermore, HOX genes of the A cluster are generally found in myelomonocytic cells, B cluster genes in erythropoietic cells, and C cluster genes in lymphoid cells. The results presented here concentrate on a single gene, namely HOX B6. Preliminary observations using whole mount in situ hybridization showed that both HOX B6 and erythropoietin (EPO) gene expression occurred in exactly the same areas of the 8.5-day mouse embryo. As a consequence, we studied the expression of HOX B6 and EPO gene expression from 6.5 to 19.5 days of gestation, in the neonate, and in the adult. It was found that the sequential transfer of erythropoiesis in different organs during development was followed by a similar transfer of HOX B6 and EPO gene expression. Between days 16.5 and 17.5, both HOX B6 and EPO gene expression decrease in the fetal liver, even though hepatic erythropoiesis continues to decline and is transferred to the fetal spleen. Precisely at this time point, HOX B6 and EPO gene expression are transferred to both the fetal spleen and fetal kidney. However, surprisingly, expression of both genes increases again in the fetal liver just before birth. HOX B6 is expressed in cells from in vitro erythropoietic colonies (colony-forming unit-erythroid and burst-forming unit-erythroid) and TER-119+ erythroid cells but not in hematopoietic or nonhematopoietic stem cell populations. When the latter two populations are allowed to differentiate into erythropoietic cells, HOX B6 and erythroid-relevant markers are expressed. The results indicate that HOX B6 is intimately involved in the regulation of the erythropoietic system and could be a marker for this lineage.
A Hematopoietic Cell L-Selectin Ligand Exhibits Sulfate-Independent Binding Activity
Authors: Sackstein Robert , Fu Ling , Allen Katrina L.
Published: October 2019

L-selectin is a leukocyte cell-surface glycoprotein that mediates adhesive interactions between circulating cells and vascular endothelium. All endothelial ligands of L-selectin characterized to date are glycoproteins that require sulfation for activity and share reactivity with MECA 79, a monoclonal antibody that recognizes a sulfate-dependent epitope involved in L-selectin attachment. We have recently identified by functional assay a glycoprotein L-selectin ligand expressed on the human hematopoietic cell line KG1a. We report here that this ligand is not recognized by MECA 79 and that it retains binding activity after metabolic inhibition of sulfation by chlorate. A native membrane L-selectin ligand exhibiting sulfate-independent function has not been described previously. Identification of this novel ligand on a nonendothelial cell type suggests that structural determinants conferring L-selectin binding may vary in a cell- and tissue-specific manner.
Thrombopoietin and Thrombin Induce Tyrosine Phosphorylation of Vav in Human Blood Platelets
Authors: Miyakawa Yoshitaka , Oda Atsushi , Druker Brian J. , Ozaki Katsutoshi , Handa Makoto , Ohashi Hideya , Ikeda Yasuo
Published: October 2019

Thrombopoietin has an essential role in megakaryopoiesis and thrombopoiesis. To investigate the signaling processes induced by thrombopoietin, we have employed human platelets and recently demonstrated that thrombopoietin induces rapid tyrosine phosphorylation of Jak-2, Tyk2, Shc, Stat3, Stat5, p120c-cbl and other proteins in human platelets. Because the apparent molecular weight of a major tyrosine phosphorylated protein in platelets stimulated by thrombopoietin is approximately 85 to 95 kD, we examined the possibility that this could be Vav, a 95-kD proto-oncogene product. Specific antisera against Vav recognized the same 95 kD protein in lysates of Jurkat cells, which are known to express Vav, and platelets, indicating that platelets have Vav. Thrombopoietin induced rapid tyrosine phosphorylation of Vav in platelets without an elevation in cytosolic free calcium concentration or activation of protein kinase C. Vav was also tyrosine phosphorylated upon treatment of platelets with thrombin, collagen, or U46619, which activate phospholipase C, leading to an increased ionized calcium concentration and activation of protein kinase C. Ionomycin or phorbol 12-myristate 13-acetate (PMA) also induces tyrosine phosphorylation of Vav, suggesting that an increase in ionized calcium concentration or activation of protein kinase C may lead to phosphorylation of Vav. Thrombopoietin also induced tyrosine phosphorylation of Vav in FDCP-2 cells, genetically engineered to express human c-Mpl (FDCP-hMpl5). However, neither ionomycin nor PMA induced an increase in tyrosine phosphorylation of Vav in FDCP-hMpl5 cells, suggesting that the calcium and protein kinase C pathways of Vav phosphorylation may be unique to platelets. Further, Vav became incorporated into the Triton X-100 insoluble 10,000g sedimentable residue in an aggregation-dependent manner, suggesting that it may have a regulatory role in platelet cytoskeletal processes. Vav was constitutively associated with a 28-kD adapter protein, Grb2, which is also incorporated into the cytoskeleton in an aggregation-dependent fashion. Lastly, we found that Vav is cleaved when there is activation of calpain, a protease that may have a role in postaggregation signaling processes. Our data suggest that thrombopoietin and other agonists may induce tyrosine phosphorylation of Vav by different mechanisms and Vav may also be involved in signaling during platelet aggregation by its redistribution to the cytoskeleton.
Absence of Linkage Between Type III Protein S Deficiency and the PROS1 and C4BP Genes in Families Carrying the Protein S Heerlen Allele
Authors: Espinosa-Parrilla Yolanda , Morell Marta , Souto Joan Carles , Borrell Montserrat , Heine-Suñer Damián , Tirado Isabel , Volpini Vı́ctor , Estivill Xavier , Sala Núria
Published: October 2019

To elucidate the molecular basis of hereditary protein S (PS) deficiency and, in particular, type III or free PS deficiency, the allelic distribution and segregation patterns of the PS gene (PROS1) polymorphisms P626A/G and S460P (PS Heerlen) have been analyzed in a group of 45 proposita suffering from type I or type III PS deficiency. No differences between patients and controls were found in the frequency of the P626A/G alleles. By contrast, the frequency of the PS Heerlen allele in the group of patients with type III PS deficiency (9 of 46 chromosomes, P = .196) was significantly higher (P < .001) than in the control group (1 of 300 chromosomes, P = .003). The A allele of P626A/G was always associated with the P allele of S460P. However, this haplotype did not cosegregate with the type III PS-deficient phenotype in 3 of the families. Furthermore, multipoint linkage analysis excluded the whole PROS1 gene in 1 of these families, which is in agreement with the absence of mutations in the PROS1 gene, as determined by sequence analysis. Finally, linkage analysis with 4 microsatellite markers linked to the C4BPB and C4BPA loci also excluded these two genes. From these results we conclude that, at least in some families, the molecular basis of type III PS deficiency is not due to the Mendelian inheritance of a single defect in the PROS1 or in the C4BP genes.
Naturally Occurring Arg−1 to His Mutation in Human Protein C Leads to Aberrant Propeptide Processing and Secretion of Dysfunctional Protein C
Authors: Lind Bent , Johnsen Anders H. , Thorsen Sixtus
Published: October 2019

The dysfunctional protein C from a thrombophilic patient heterozygote for a G1388 to A converting the codon for Arg−1 to His was purified from plasma and characterized. N-terminal amino acid sequence analysis of the light chain of the protein C demonstrated that the dysfunctional protein C is elongated with one amino acid, namely the mutated His. This finding is compatible with disruption by the mutated His of the original basic propeptidase recognition sequence (Arg−5-Ile-Arg-Lys-Arg−1), resulting in a shift of the cleavage site to a new position, Lys−2-His−1, which follows an alternative basic amino acid propeptidase recognition sequence (Arg−5-Ile-Arg-Lys−2). Because the mutation affects the propeptide that directs the γ-carboxylation converting Glu to Gla residues in the Gla domain, it was investigated whether the mutation impaired this reaction. Gla fragment obtained by cleavage of the dysfunctional protein C light chain with endoproteinase Asp-N was isolated by reverse-phase high-performance liquid chromatography, methylated, and subjected to N-terminal sequence analysis. The methylation step enabled the positive identification of Gla residues as well as the determination of the relative amount of Gla and Glu residues at each of the nine γ-carboxylation sites of the Gla domain. The analysis showed that all nine potential γ-carboxylation sites of the dysfunctional protein C were normally carboxylated. This result is compatible with the notion that position −1 is not a part of the recognition element for the γ-carboxylase. In conclusion, evidence is provided showing that the mutation leads to aberrant propeptide processing and secretion of dysfunctional normally carboxylated protein C extended with the mutated His.
Two Distinct Pathways of Interleukin-5 Synthesis in Allergen-Specific Human T-Cell Clones Are Suppressed by Glucocorticoids
Authors: Mori Akio , Kaminuma Osamu , Suko Matsunobu , Inoue Satoshi , Ohmura Takeo , Hoshino Akihiko , Asakura Yumiko , Miyazawa Keiji , Yokota Toyokazu , Okumura Yasushi , Ito Koji , Okudaira Hirokazu
Published: October 2019

Glucocorticoids (GC) have long been used as the most effective agents for the treatment of allergic diseases accompanied by eosinophilia such as chronic asthma and atopic dermatitis. The development of chronic eosinophilic inflammation is dependent on interleukin-5 (IL-5), a selective eosinophil-activating factor, produced by helper T cells. To delineate the regulatory mechanisms of human IL-5 synthesis, we established allergen-specific CD4+ T-cell clones from asthmatic patients. GC efficiently suppressed IL-5 synthesis of T-cell clones activated via either T-cell receptor (TCR) or IL-2 receptor (IL-2R). Induction of IL-5 mRNA upon TCR and IL-2R stimulation was totally inhibited by dexamethasone. Human IL-5 promoter/enhancer-luciferase gene construct transfected to T-cell clones was transcribed on either TCR or IL-2R stimulation and was clearly downregulated by dexamethasone, indicating that the approximately 500-bp human IL-5 gene segment located 5′ upstream of the coding region contains activation-inducible enhancer elements responsible for the regulation by GC. Electrophoretic mobility shift assay analysis suggested that AP-1 and NF-κB are among the possible targets of GC actions on TCR-stimulated T cells. NF-AT and NF-κB were not significantly induced by IL-2 stimulation. Our results showing that GC suppressed IL-5 production by human CD4+ T cells activated by two distinct stimuli, TCR and IL-2R stimulation, underscore the efficacy of GC in the treatment of allergic diseases via suppression of T-cell IL-5 synthesis.
Altered Expression of the Retinoblastoma Tumor-Suppressor Gene in Leukemic Cell Lines Inhibits Induction of Differentiation But Not G1-Accumulation
Authors: Bergh Gösta , Ehinger Mats , Olofsson Tor , Baldetorp Bo , Johnsson Ellinor , Brycke Helena , Lindgren Gustaf , Olsson Inge , Gullberg Urban
Published: October 2019

The retinoblastoma tumor-suppressor gene, RB, has been implicated in tumor suppression, in regulation of the cell cycle, and in mediating cell differentiation. RB is necessary for hematopoiesis in mice, and aberrant RB-expression is associated with the progress and prognosis of leukemia. We have used antisense oligonucleotides, established clones stably expressing an antisense RB construct, and also established clones over expressing the retinoblastoma protein (pRb) to study the role of RB expression in monocytic differentiation induced by all-trans retinoic acid (ATRA) or 1-α-25-dihyroxycholecalciferol (Vit D3) in the monoblastic cell line U-937 and erythroid differentiation induced by transforming growth factor β1 (TGFβ1) and hemin in the erythroleukemic cell line K562. A reduction in pRb production in antisense RB-transfected U-937 clones was shown. Antisense oligonucleotides as well as expression of the antisense RB construct suppressed differentiation responses to ATRA or Vit D3, as judged by the capability to reduce nitro blue tetrazolium, by the appearance of monocyte-related cell surface antigens and by morphologic criteria. K562 cells showed decreased differentiation response to TGFβ1, but not to hemin, when incubated with antisense oligonucleotides. U-937 antisense RB-transfected cells were also suppressed in their ability to upregulate levels of hypophosphorylated pRb when induced to differentiate. Although U-937 cells incubated with antisense oligonucleotides and clones expressing the antisense RB construct were hampered in their ability to differentiate on incubation with ATRA or Vit D3, the induced G0/G1-accumulation was similar to differentiating control cells treated with ATRA or Vit D3. Intriguingly, U-937 clones overexpressing RB were also inhibited in their differentiation response to ATRA or Vit D3 but not inhibited in their ability to respond with G0/G1 accumulation when induced with these substances. The results indicate that pRb plays a role in induced differentiation of U-937 cells as well as K562 cells involving mechanisms that, at least partially, are distinct from those inducing G1 accumulation.
The Expression of Bcl-x Is Downregulated During Differentiation of Human Hematopoietic Progenitor Cells Along the Granulocyte But Not the Monocyte/Macrophage Lineage
Authors: Sanz Cristina , Benito Adalberto , Silva Maite , Albella Beatriz , Richard Carlos , Segovia Jose Carlos , Insunza Andres , Bueren Juan Antonio , Fernández-Luna Jose Luis
Published: October 2019

Expression of the apoptosis inhibitory protein Bcl-x was studied in CD34+ hematopoietic precursor cells and in the promyelocytic leukemia cell line HL-60. The enriched population of CD34+ cells (more than 95%) was cultured in the presence of stem cell factor, interleukin-3 (IL-3), IL-6, and either granulocyte colony-stimulating factor or macrophage colony-stimulating factor to achieve granulocyte or monocyte/macrophage differentiation, respectively. The expression of Bcl-x increased in the early stages of both differentiation pathways. However, by day 21 of culture mature granulocytes were Bcl-x–negative, whereas monocytes/macrophages either maintained or increased the expression of Bcl-x. The pattern of Bcl-x expression in the differentiated CD34+ cells was similar to that observed in HL-60 cells differentiated along the granulocyte lineage (induced by incubation with retinoic acid), or along the monocyte/macrophage lineage (induced by incubation with phorbol diester). The bcl-x transcript predominant in HL-60 and CD34+ cells differentiated into monocytes/macrophages was bcl-xL . Although little is yet known regarding the functional significance of Bcl-x within the granulomonocytic compartment, marked changes in the pattern of its expression, as observed during granulomonocytic differentiation of HL-60 and CD34+ cells, are likely to alter the life span of mature granulocytes and monocytes/macrophages.
A Soluble Tissue Factor Mutant Is a Selective Anticoagulant and Antithrombotic Agent
Authors: Kelley Robert F. , Refino Canio J. , O'Connell Mark P. , Modi Nishit , Sehl Pat , Lowe David , Pater Cheryl , Bunting Stuart
Published: October 2019

One approach to developing safer and more efficacious agents for the treatment of thrombotic disease involves the design and testing of inhibitors that block specific steps in the coagulation cascade. We describe here the development of a mutant of human tissue factor (TF ) as a specific antagonist of the extrinsic pathway of blood coagulation and the testing of this mutant in a rabbit model of arterial thrombosis. Alanine substitutions of Lys residues 165 and 166 in human TF have been shown previously to diminish the cofactor function of TF in support of factor X (FX) activation catalyzed by factor VIIa (FVIIa). The K165A:K166A mutations have been incorporated into soluble TF (sTF; residues 1-219) to generate the molecule “hTFAA.” hTFAA binds FVIIa with kinetics and affinity equivalent to wild-type sTF, but the hTFAA⋅FVIIa complex shows a 34-fold reduction in catalytic efficiency for FX activation relative to the activity measured for sTF⋅FVIIa. hTFAA inhibits the activation of FX catalyzed by the complex formed between FVIIa and relipidated TF(1-243). hTFAA prolongs prothrombin time (PT) determined with human plasma and relipidated TF(1-243) or membrane bound TF, and has no effect on activated partial thromboplastin time, but is 70-fold less potent as an inhibitor of PT with rabbit plasma. The rabbit homologue of this mutant (“rTFAA”) was produced and shown to have greater potency with rabbit plasma. Both hTFAA and rTFAA display an antithrombotic effect in a rabbit model of arterial thrombosis with rTFAA giving full efficacy at a lower dose than hTFAA. Compared to heparin doses of equal antithrombotic potential, hTFAA and rTFAA cause less bleeding as judged by measurements of the cuticle bleeding time. These results indicate that TF⋅FVIIa is a good target for the development of new anticoagulant drugs for the treatment of thrombotic disease.
Interleukin-4 Induces Homotypic Aggregation of Human Mast Cells by Promoting LFA-1/ICAM-1 Adhesion Molecules
Authors: Toru Hano , Kinashi Tatsuo , Ra Chisei , Nonoyama Shigeaki , Yata Jun-ichi , Nakahata Tatsutoshi
Published: October 2019

We report here that interleukin-4 (IL-4) induces homotypic aggregation of cultured human mast cells, grown from cord blood mononuclear cells in the presence of stem cell factor and IL-6. This aggregation was specifically induced by IL-4, because other cytokines including IL-1α, IL-1β, IL-2, IL-3, IL-5, IL-9, IL-10, interferon-γ, IL-12, granulocyte-macrophage colony-stimulating factor, NGF-β, and tumor necrosis factor-α failed to show such effect. Flow cytometric analysis of the cultured mast cells showed that IL-4 increases the expression of lymphocyte function-associated antigen-1 (LFA-1) and intercellular adhesion molecule-1 (ICAM-1), but not of very late antigen (VLA) family adhesion molecules or vascular cell adhesion molecule-1 (VCAM-1). Neutralizing monoclonal antibodies specific for LFA-1α, LFA-1β, or ICAM-1 inhibited the IL-4–induced homotypic aggregation of the mast cells, indicating that the aggregation was mediated mainly by LFA-1/ICAM-1 interaction. In addition, IL-4–treated but not untreated mast cells bound to immobilized ICAM-1. This binding was also inhibited by anti-LFA-1 or anti-ICAM-1. These results show that IL-4 promotes expression of ICAM-1 and LFA-1 molecules on mast cells, and suggest that IL-4 may contribute to the migration of mast cells into the inflamed tissue and to the cellular interaction with other inflammatory cells by upregulating adhesion molecules.
Dose-Dependent Enhancements by Interferon-γ on Functional Responses of Neutrophils From Chronic Granulomatous Disease Patients
Authors: Åhlin Anders , Elinder Göran , Palmblad Jan
Published: October 2019

Interferon-γ (IFN-γ) is recommended as prophylaxis against infections in patients with chronic granulomatous disease (CGD). However, since the optimal dose, the dosing interval, and the mechanisms of action are not well-defined, we studied the effects on CGD neutrophil (PMN) functions ex vivo of interferon-γ (IFN-γ). Evaluations were made on oxidative capacity, measured by superoxide anion production and chemiluminescence after stimulation with f-met-leu-phe (f-MLP) or phorbol-myristate-acetate, the killing of Aspergillus fumigatus hyphae (assessed as conversion of the tetrazolium salt MTT to formazan), and on the expression of FcγRI receptor (CD64). After randomization, 9 CGD patients (4 with gp91phox, 3 with p47phox, 1 with p67phox deficiency and 1 with unspecified CGD) were given IFN-γ, either 50 or 100 μg/m2 subcutaneously on 2 consecutive days after double blinded randomization. Furthermore, one female hyperlyonized X-linked carrier with a CGD phenotype was also studied separately after IFN-γ treatment. Evaluations were made the day before and on days 1, 3, 8, and 18 after IFN-γ administration. The killing of A fumigatus hyphae, being close to zero before IFN-γ, was enhanced on day 3, being 36% higher than pretreatment values in the high-dose CGD group and 17% in the low-dose group. The expression of FcγRI on PMN increased 3.7-fold in the high-dose and 2.3-fold in the low-dose CGD group, being maximal on day 1. Oxidative functions were raised in only selected patients represented by different subtypes of CGD. The hyperlyonized carrier of X-linked CGD responded to IFN-γ with more enhanced oxidative responses and Aspergillus killing of her PMNs than the other patients. This study suggests that a higher dose of IFN-γ than currently recommended confers transient enhancements of certain PMN functions in CGD patients.
The Antineoplastic Agent Bryostatin-1 Induces Proinflammatory Cytokine Production in Human Monocytes: Synergy With Interleukin-2 and Modulation of Interleukin-2Rγ Chain Expression
Authors: Bosco Maria Carla , Rottschafer Sandra , Taylor Lynn S. , Ortaldo John R. , Longo Dan L. , Espinoza-Delgado Igor
Published: October 2019

The antineoplastic agent bryostatin-1 (bryo-1) possesses powerful immunomodulatory properties and can function as a biological response modifier in vivo. However, there is currently little information regarding the effects of bryo-1 on cells of the monocytic lineage. In this study, we demonstrate that bryo-1 can potently induce the production of proinflammatory cytokines from human peripheral blood monocytes. Stimulation of monocytes with subnanomolar concentrations of bryo-1 significantly upregulated the constitutive levels of interleukin-8 (IL-8) mRNA and induced the expression of IL-1β, tumor necrosis factor-α (TNF-α), and IL-6 mRNA in a time and dose-dependent manner. Accordingly, secretion of all four proinflammatory cytokines was induced after monocyte exposure to bryo-1. Furthermore, we showed that bryo-1 selectively synergized with IL-2 in triggering monocyte activation, and this effect seemed to be dependent, at least in part, on the ability of bryo-1 to upregulate IL-2Rγ chain expression. Finally, we demonstrated that the responses of monocytes to bryo-1 could be blocked by the protein kinase C (PKC) inhibitors staurosporine and UCN-01, indicating a role for PKC in monocyte activation by bryo-1. These results show for the first time that bryo-1 is a powerful activator of human monocytes and suggest that stimulation of monokine secretion by bryo-1 may represent at least one of the mechanisms responsible for the in vivo antitumor activity of this drug.
Detection and Isolation of Gene-Corrected Cells in Gaucher Disease Via a Fluorescence-Activated Cell Sorter Assay for Lysosomal Glucocerebrosidase Activity
Authors: Lorincz Matthew , Herzenberg Leonard A. , Diwu Zhenjun , Barranger John A. , Kerr William G.
Published: October 2019

Gaucher disease type 1 results from the accumulation of glucocerebroside in macrophages of the reticuloendothelial system, as a consequence of a deficiency in glucocerebrosidase (GC) activity. Recent improvements in the methodologies for introducing foreign genes into bone marrow stem cells have prompted several groups to test the efficacy of gene transfer therapy as a curative treatment for Gaucher disease. Limitations of this approach include the potential for insufficient engraftment of gene-corrected cells and incomplete transduction of hematopoietic stem cells using retroviral gene transfer. Overcoming these obstacles may be critical in the case of treatment for Gaucher disease type 1, because GC transduced cells have not been shown to have a growth advantage over noncorrected cells. Here, we describe the development and application of a novel, fluorescence-activated cell sorter based assay that directly quantitates GC activity at the single cell level. In a test of this application, fibroblasts from a Gaucher patient were transduced, and high expressing cells sorted based on GC activity. Reanalysis of cultured sorted fibroblasts reveals that these cells maintain high levels of enzymatic activity, compared with the heterogeneous population from which they were sorted. The assay is sufficiently sensitive to distinguish GC activity found in Gaucher patient monocytes from that in normal controls. Furthermore, preliminary results indicate that increased GC activity can be detected in transduced, CD34+ enriched peripheral blood mononuclear cells isolated from a Gaucher patient. This method should be a useful addition to current gene therapy protocols as a means to quantitatively assess gene correction of relevant cell populations and potentially purify transduced cells for transplantation.
Phylogenetic Footprinting of Hypersensitive Site 3 of the β-Globin Locus Control Region
Authors: Shelton David A. , Stegman Lauren , Hardison Ross , Miller Webb , Bock Jeffery H. , Slightom Jerry L. , Goodman Morris , Gumucio Deborah L.
Published: October 2019

Hypersensitive site 3 (HS3) of the β-like globin locus control region has been implicated as an important regulator of the β-like globin genes, but the trans factors that bind HS3 have only been partially characterized. Using a five-species alignment (human, galago, rabbit, goat, and mouse) that represents 370 million years of evolution, we have identified 24 phylogenetic footprints in the HS3 core and surrounding regions. Probes corresponding to the human sequence at each footprint have been used in binding studies to identify the nuclear factors that bind within and near these conserved sequence elements. Among the high-affinity interactions observed were several binding sites for proteins with repressor activity, including YY1, CCAAT displacement protein, and G1/G2 complexes (uncharacterized putative repressors) and several binding sites for the stage selector protein. To complement this analysis, orthologous galago sequences were also used to derive probes and the pattern of proteins binding to human and galago probes was compared. Binding interactions differing between these two species could be responsible for the different expression patterns shown by the two γ genes (galago γ is embryonic; human γ is fetal). Alternatively, binding interactions that are conserved in the two species may be important in the regulation of common expression patterns (eg, repression of γ in adult life).
A Patient With Paroxysmal Nocturnal Hemoglobinuria Bearing Four Independent PIG-A Mutant Clones
Authors: Nishimura Jun-ichi , Inoue Norimitsu , Wada Hiroshi , Ueda Etsuko , Pramoonjago Patcharin , Hirota Toshiyuki , Machii Takashi , Kageyama Takashi , Kanamaru Akihisa , Takeda Junji , Kinoshita Taroh , Kitani Teruo
Published: October 2019

Paroxysmal nocturnal hemoglobinuria (PNH) is characterized by clonal blood cells that are deficient in the surface expression of glycosylphosphatidylinositol-anchored proteins due to somatic mutation in the X-linked gene PIG-A. In some patients, more than one abnormal clone may be present. Analysis of bulk DNA/RNA from granulocytes has been useful in identifying the predominant PIG-A mutation in each patient. However, it is often not useful in determining the presence of minor clones. Many patients have cells with partial deficiency. Here, we analyzed the PIG-A gene in two B-cell lines bearing complete or partial deficiencies, cells of hematopoietic progenitor colonies and peripheral blood granulocytes from the same patient. We found that two B-cell lines had different mutations, the granulocytes contained at least two mutants, and the hematopoietic progenitors contained four mutants. Three of the four were shared by B cells and/or granulocytes whereas the other one was found only in the hematopoietic progenitors. The partial deficiency was caused by a point mutation near an alternative splice site within exon 2 that resulted in partial decreases of activity and quantity of the full-length transcript. These results further show the oligoclonal nature of PNH and differences in extent of expansion among mutant clones.
The Role of B7 Costimulation by Murine Acute Myeloid Leukemia in the Generation and Function of a CD8+ T-Cell Line With Potent In Vivo Graft-Versus-Leukemia Properties
Authors: Boyer Michael W. , Vallera Daniel A. , Taylor Patricia A. , Gray Gary S. , Katsanis Emmanuel , Gorden Keith , Orchard Paul J. , Blazar Bruce R.
Published: October 2019

Relapse is more frequent after autologous than allogeneic bone marrow transplantation (BMT), due in part to lack of T-lymphocyte mediated allogeneic graft-versus-leukemia (GVL) effects. Infusions of leukemia-reactive T cells to patients after autologous BMT may be a means for providing a GVL effect. Costimulation of T cells by binding of the CD28 receptor on T cells with B7-counter receptors on antigen presenting cells amplifies antigen-specific T-cell responses. To enhance generation of leukemia reactive cytotoxic T lymphocytes (CTL), the murine B7-1– and B7-2–costimulatory molecule cDNAs were introduced into the MHC class I+, class II−, murine meyloid leukemia cell line C1498. B7-1 expression greatly enhanced the ability of the leukemia cells to generate and expand leukemia reactive CTL in vitro. A highly cytolytic and C1498 specific CD8+ CTL line was generated by B7-1 costimulation. This CTL line proliferated autonomously and produced interleukin-2 when provided B7-1 or B7-2 costimulation by C1498 leukemia cells. To test the in vivo antileukemia properties of this CTL line, irradiated syngeneic BMT recipients were given graded doses of leukemia cells on day 0, followed by CTL infusions beginning on day 1 post-BMT. Recipients of 107 CTL had a 3 log reduction in leukemia burden such that 100% of mice were protected from a supralethal leukemic cell dose. Sustained immune responses were detectable up to 3 months postinfusion of the CTL line. B7-1 or B7-2 costimulation in vivo did not augment antileukemia effects of infused CTL post BMT. These results suggest that B7 costimulation of leukemia reactive CTL may be important for their ex vivo generation and expansion for use in human adoptive immunotherapy of leukemia.
New Observations on Sickle Cells
Authors: BESSIS M. , BRICKA M. , BRETON-GORIUS J. , TABUIS J.
Published: October 2019

Observations made on sickle cells with phase contrast and with the electron microscope lead to the following conclusions.

1. When the red cell sickles, its oscillatory movements cease.

2. Spherical red cells give rise to a special form of sickle cell.

3. Myelin forms, whether attached to the cell or free, become rigid when the tension of oxygen is reduced.

4. The electron microscope, combined with the technic of moulage, shows that the sickle cell contains striated rod-like structures, which give a characteristic appearance to the surface of the sickle cell.

5. Agglutination by appropriate antisera does not occur to any extent in sickled cells, because these rigid structures do not form myelin forms. This is additional evidence of the importance of the formation of viscous surface layers already described as part of the mechanism of agglutination.
The Simultaneous Occurrence of Benign Thymoma and Refractory Anemia
Authors: ROSS JOSEPH F. , FINCH STUART C. , STREET RUSSELL B. , STRIEDER JOHN W.
Published: October 2019

1. The simultaneous occurrence of a benign thymoma and a severe anemia which failed to respond to therapy with recognized hematopoietic agents has been observed in two adult female patients.

2. Reports of seven other patients with a similar association of thymoma and refractory anemia have been found and the, salient features of these cases are summarized.

3. Possible explanations of the simultaneous occurrence of these two unusual conditions are discussed.

4. It is suggested that the thymus gland or tumors of the thymus may be etiologically responsible for certain cases of refractory anemia.
Chronic Acquired Hemolytic Anemia Associated with Hemoglobinuria and Raynaud's Phenomena
Authors: BONNIN J. A.
Published: October 2019

The clinical history and serological findings in a patient suffering from chronic hemolytic anemia with Raynaud's phenomena and hemoglobinuria are briefly reported. The patient's serum contained a cold antibody in very high concentration, which was active up to a temperature of at least 33.5 C. Its activity in vitro differed from that of the sera of previously reported cases in that the hemolysis of normal red cells was diminished rather than increased by lowering the pH. It is thought probable that the patient's frequent attacks of hemoglobinuria may be correlated with the ease with which hemolysis took place in vitro at the physiological pH.
Studies on Abnormal Hemoglobins
Authors: SINGER KARL , KRAUS ALFRED P. , SINGER LILY , RUBINSTEIN HERBERT M. , GOLDBERG SEYMOUR R.
Published: October 2019

1. A Negro family is described in which several individuals exhibit either the manifestations of thalassemia, or of the uncomplicated hemoglobin C trait; in two members the combined occurrence of these two anomalies is demonstrable. This combination is designated as hemoglobin C—thalassemia disease.

2. Hemoglobin C—thalassemia disease manifests itself in these two patients as a microcytic erythrocytosis. The red cells reveal a low MCV and low MCH, but a normal MCHC. About 45 per cent of the erythrocytes appear as target cells in the film. The amount of hemoglobin C in the hemolysates was about 75 per cent, the remainder being composed of A hemoglobin, and in one instance also of a small quantity of F hemoglobin. This is analogous to the results of the hemoglobin analyses in sickle cell—thalassemia disease, where 60 to 80 per cent of the pathologic hemoglobin S are found, though these individuals are heterozygous for both the pathologic hemoglobin and the thalassemia genes. The hypothesis is advanced that the thalassemia gene modifies (enhances) the expressivity of the gene for the pathologic pigment.

3. In genetic studies of families with thalassemia, hemoglobin analyses represent a necessary requirement. It is now established that thalassemia, as well as disorders associated with hemoglobin C, reveals a tendency to leptocytosis and thus may show erythrocytes with increased osmotic resistance.

4. The segregation of the thalassemia gene and of the genes for pathologic hemoglobins take place independently of each other. These genes are not allelomorphs.