bluuebunny/crossref_metadata_2025 huggingface dataset:
Comparative studies of microscopically determined aggregation, degranulation, and light transmission after chemotactic activation of adult and newborn neutrophils
Authors: Rochon YP , Frojmovic MM , Mills EL
Published: October 2019

Changes in the light transmission of suspensions of activated neutrophils are widely used to measure the dynamics of neutrophil aggregation. Such studies have suggested, for example, that aggregation is irreversible for human newborn neutrophils but fully reversible for adult cells. We have evaluated aggregation directly by microscopic particle counting and compared it with changes in light transmission (delta T) and with release from three granule subsets for neutrophils activated with N-formyl-methionyl-leucyl-phenylalanine (FMLP). Maximal increases in %T in response to 0.5 micromol/L FMLP were approximately 25% larger for newborn than for adult neutrophils, and were only partially reversible by 8 minutes, while %T increases for adult neutrophils were fully reversible. However, measurements of neutrophil aggregation using light microscopy showed that both newborn and adult neutrophils fully deaggregated. A further independence of delta T from aggregation was found by pretreating adult neutrophils with cytochalasin B (5 micrograms/mL) in the presence of 0.5% gelatin, a pretreatment that blocked FMLP-induced neutrophil aggregation while allowing large increases in %T and degranulation. In response to FMLP, newborn neutrophils released more enzyme from each granule subset than did adult neutrophils. Our results suggest that cellular events associated with neutrophil activation (other than aggregation) are implicated in light transmission responses and that these differ for adults and newborns. These results also suggest that reports of neutrophil aggregation should be based on direct particle counting methods rather than on %T responses.
Reconstitution of T-cell function after CD6-depleted allogeneic bone marrow transplantation
Authors: Soiffer RJ , Bosserman L , Murray C , Cochran K , Daley J , Ritz J
Published: October 2019

Patients who undergo allogeneic bone marrow transplantation (BMT) are clinically immunodeficient for a prolonged period after engraftment. In the present study, we examined immune function after BMT in a series of patients who had received HLA compatible sibling marrow grafts purged of T cells with anti-CD6 monoclonal antibody and complement. None of the patients in this analysis received immunomodulating agents and none had developed graft-versus-host disease (GVHD). Initially after BMT, natural killer (NK) cells are the predominant cell type, giving way to CD3+, CD5+ T cells after 4 to 8 weeks. Despite the return of normal numbers of T lymphocytes post-BMT phenotypic analysis reveals several long-term abnormalities, including an inverted T4:T8 ratio and a significant fraction of CD3+ T cells that do not co-express CD6. In mitogenic assays, stimulation by either nonspecific lectin (phytohemagglutinin; PHA) or antibodies to the CD2 surface structure (anti-T11(2) + anti-T11(3)) results in decreased levels of T-cell proliferation compared with controls for over 18 months post-BMT. In contrast, the ability of unstimulated peripheral blood mononuclear cells (PBMC) to respond to recombinant interleukin-2 (rIL-2) is relatively intact, most likely reflecting early functional reconstitution of the NK cell population. To further characterize the prolonged abnormalities in T-cell proliferation after PHA or CD2 stimulation, we examined more proximal events in T-cell activation such as induction of IL-2 receptor expression and stimulus-induced intracellular calcium flux. We found that the induction of IL-2 receptor (p55) after in vitro activation, although initially abnormal, recovers completely by 6 months post-BMT. We also found that, after CD2 stimulation, calcium flux in T cells was normal immediately after engraftment. In contrast, after stimulation with anti-CD3 antibodies, a large population of T cells do not develop intracellular calcium flux compared with controls. We conclude that despite the recovery of normal numbers of T lymphocytes early after engraftment of CD6-depleted marrow, these T cells exhibit several physiologic and functional abnormalities that persist for varying intervals post-BMT. At present, it is unclear which of these specific defects is most closely associated with increased susceptibility to infectious agents after BMT.
Expression of shared idiotypes by paraproteins from patients with multiple myeloma and monoclonal gammopathy of undetermined significance
Authors: Berenson JR , Lichtenstein A , Hart S , Palomares D , Miller RA
Published: October 2019

Twenty-nine murine monoclonal antibodies have been produced that react with shared idiotypes expressed by B-cell lymphomas and leukemias. We tested this panel of antibodies for reactivity with the paraproteins from 32 patients with multiple myeloma and 10 patients with monoclonal gammopathy of undetermined significance (MGUS). Thirteen of 42 paraproteins reacted with at least one antibody in this panel of anti- idiotypic antibodies. Six different anti-idiotypes demonstrated reactivity with the paraproteins. A similar frequency of reactivity was found for both myeloma and MGUS proteins. One antibody, S30–47, reacted with 6 of 32 (19%) of the paraproteins from patients with multiple myeloma, whereas this anti-idiotype only bound to 3% of non-Hodgkin's B- cell lymphomas and no cases of chronic lymphocytic leukemia. This anti- idiotype reacted with both components of a biphenotypic paraprotein (IgG kappa and IgG lambda) in one patient. In each of nine patients tested, plasma cells isolated from bone marrow were shown to be reactive with the same anti-idiotype we found to react with the paraprotein. Antishared idiotype antibodies may provide useful reagents for studies of patients with monoclonal gammopathies.
Structural and functional heterogeneity of alpha spectrin mutations involving the spectrin heterodimer self-association site: relationships to hematologic expression of homozygous hereditary elliptocytosis and hereditary pyropoikilocytosis
Authors: Coetzer T , Palek J , Lawler J , Liu SC , Jarolim P , Lahav M , Prchal JT , Wang W , Alter BP , Schewitz G
Published: October 2019

Defects involving alpha spectrin (Sp) are found in patients with hereditary elliptocytosis and a related disorder, hereditary pyropoikilocytosis (HPP). We have previously found that the severity of hemolysis was related to the total spectrin content of the cells and the percentage of unassembled dimeric Sp (SpD) in the membranes, which, in turn, reflected the amount of mutant Sp in the cell. However, no data are available comparing differences in the function of various alpha Sp mutations to clinical severity. We now report studies of nine homozygotes or double heterozygotes for four alpha Sp mutations: alpha 1/74, alpha 1/46, alpha 1/65, and alpha 1/61, whose red blood cells (RBCs) contained only the mutant Sp and no normal Sp. Sp alpha 1/74, Sp alpha 1/46, and alpha 1/65 homozygotes differed strikingly in the severity of hemolysis that correlated with the severity of mutant Sp dysfunction, as reflected by the fraction of unassembled SpD in the membranes and the self-association of mutant Sp on inside-out vesicles. Homozygotes for Sp alpha 1/74 had a very severe hemolytic anemia and their SpD were virtually incapable of self-association, whereas SpD alpha 1/46 were not as severely affected. The Sp alpha 1/65 homozygotes had a relatively mild hemolytic anemia and their SpD showed the least impairment of function. Ultrastructural examination of membrane skeletons from subjects whose SpD self-association was severely impaired showed gross skeletal disruption and loss of hexagonal structure. In striking contrast, the homozygote for the mildly dysfunctional Sp alpha 1/65 had only a moderate disruption of the skeleton. Some of the homozygous or doubly heterozygous subjects also exhibited a partial deficiency of Sp that correlated with a RBC morphology characteristic of HPP, namely, marked microspherocytosis with virtual absence of elliptocytes. These data demonstrate striking differences in the function and structure of various alpha Sp mutants that underlie differences in clinical expression.
Regulation of the gene for CD34, a human hematopoietic stem cell antigen, in KG-1 cells
Authors: Satterthwaite AB , Borson R , Tenen DG
Published: October 2019

CD34 is one of the best characterized human hematopoietic stem cell antigens defined to date. It is expressed on 1% to 4% of normal bone marrow cells, including colony forming units of all lineages and their precursors. CD34 expression is lost during hematopoietic development and is not found on mature peripheral blood cells. The control of CD34 expression was studied in the myeloblast cell line KG-1 as a model for the regulation of stem cell genes. CD34 mRNA was expressed at high levels in uninduced KG-1 cells. Upon induction of the cells towards macrophages with tetradecanoylphorbol-13-acetate (TPA) and ionomycin, steady state levels of CD34 mRNA decreased rapidly. Nuclear run-on assays did not show a significant change in the rate of CD34 transcription upon induction. The half-life of CD34 mRNA was 4.5 hours in uninduced KG-1 cells and 2.25 hours in induced cells, demonstrating the involvement of post-transcriptional mechanisms in CD34 downregulation. Cycloheximide had no effect on the downregulation of CD34, suggesting that labile proteins are not required for this process. This model should allow the study of some of the regulatory mechanisms controlling early events in hematopoiesis.
Polarized fibronectin secretion and localized matrix assembly sites correlate with subendothelial matrix formation
Authors: Kowalczyk AP , Tulloh RH , McKeown-Longo PJ
Published: October 2019

Endothelial cells in vivo form the interface between the vascular and interstitial compartments and are strategically located to mediate vascular permeability and hemostasis. One mechanism endothelial cells use to maintain a nonthrombogenic surface is to polarize basement membrane constituents to the basolateral cell surface. In the present study, we began characterization of the mechanisms used by endothelial cells in the assembly of a subcellular fibronectin matrix. Immunofluorescence microscopy was used to localize extracellular matrix fibronectin in endothelial cell cultures. In contrast to preconfluent and newly confluent cultures, post-confluent cultures assembled a fibronectin matrix that was restricted to the basolateral cell surface. To determine if endothelial cells polarize fibronectin secretion, Millicell culture inserts were used to distinguish proteins secreted from apical and basal surfaces. Preconfluent and newly confluent cultures secreted fibronectin equally into apical and basal media. In contrast, post-confluent endothelial cells secreted fibronectin preferentially into the basal chamber. The degree to which fibronectin secretion was polarized varied with time in culture and with the ability of the monolayers to act as a barrier to the movement of 125I- fibronectin from the apical to basal chamber. In addition, high affinity binding sites for exogenous 125I-fibronectin were found to be present on the basolateral, but not apical, surface of post-confluent endothelial monolayers. These results indicate that subendothelial matrix assembly correlates with polarized fibronectin secretion, culture confluence, and expression of high affinity binding sites for fibronectin on the basolateral cell surface.
Production of monoclonal antibodies that detect Hodgkin's high molecular weight transforming growth factor-beta
Authors: Newcom SR , Muth LH , Parker ET
Published: October 2019

High molecular weight transforming growth factor-beta (TGF beta) is a physiologically active TGF secreted by nodular sclerosing Reed- Sternberg cells. Five monoclonal murine antibodies were prepared that distinguished Hodgkin's TGF beta from platelet-derived TGF beta using an enzyme-linked immunosorbent assay, neutralization of biologic activity, and Western blotting. These monoclonal antibodies directed at unique antigenic determinants (epitopes) of Hodgkin's TGF beta will allow further characterization of the role of Hodgkin's TGF beta in Hodgkin's disease and related entities.
Tyrosine-specific protein phosphorylation during activation of human neutrophils
Authors: Berkow RL , Dodson RW
Published: October 2019

The activation of human neutrophils by a variety of receptor-dependent and receptor-independent agonists induces the phosphorylation of a large number of proteins. Since we have previously shown that human neutrophils have at least two distinct tyrosine kinase activities, we examined protein tyrosine phosphorylation in human neutrophils stimulated with a variety of agonists. Using a monoclonal antibody specific for phosphotyrosine, the present study shows that the chemotactic peptides FMLP and leukotriene B4, the phorbol ester phorbol myristate acetate (PMA), and the calcium ionophore A23187 induce an increase in tyrosine phosphorylation of a number of neutrophil proteins. This increased protein tyrosine phosphorylation was dependent on the concentration of the agonist, as well as on the time of exposure to the agonist. Fractionation experiments showed that both a 150,000 g cytosolic and a particulate preparation showed increases in protein tyrosine phosphorylation with stimulation by FMLP or PMA, and showed that the pattern of protein tyrosine phosphorylation was slightly different in the FMLP- and PMA-stimulated cells. These data indicate that protein tyrosine phosphorylation is an early event in the activation of human neutrophils by a variety of receptor-dependent and receptor-independent agonists.
Serial transplantation of methotrexate-resistant bone marrow: protection of murine recipients from drug toxicity by progeny of transduced stem cells
Authors: Corey CA , DeSilva AD , Holland CA , Williams DA
Published: October 2019

Recombinant retroviral vectors have been used to transfer a variety of genetic sequences into hematopoietic stem cells. Although transfer and expression of foreign genetic sequences into reconstituting stem cells is one approach to somatic gene therapy, few studies have shown long lasting phenotypic changes in recipient mice in vivo. In this study, we show successful transfer of a methotrexate-resistant cDNA (DHFRr) into reconstituting hematopoietic stem cells using a retroviral vector, FrDHFRr, in which the DHFR cDNA is expressed off a hybrid Friend/Moloney long term repeat. Both primary and secondary recipients transplanted with bone marrow cells infected with this recombinant retrovirus show improved survival and protection from methotrexate- induced marrow toxicity when compared with control animals. These data suggest that retroviral-mediated gene transfer of DHFRr cDNA leads to a stable change in the phenotype of hematopoietic stem cells and progeny derived from those cells in vivo after bone marrow transplantation. Gene transfer using recombinant retroviral vectors seems to be one rational approach to establishing chemotherapy-resistant bone marrow cells.
A de novo and heterozygous gene deletion causing a variant of von Willebrand disease
Authors: Bernardi F , Marchetti G , Guerra S , Casonato A , Gemmati D , Patracchini P , Ballerini G , Conconi F
Published: October 2019

An abnormal von Willebrand factor (vWF) gene restriction pattern has been found in a patient with von Willebrand disease. Because this gene alteration is not present in his parents or in 50 normal and 25 affected subjects, and the restriction fragment length polymorphism haplotypes are inherited normally in the patient's family, we suggest that a de novo mutation is present. Bands with reduced intensity and additional fragments, observed in several restriction digests, hybridize with noncontiguous copy DNA (cDNA) portions, thus indicating the presence of a heterozygous gene deletion. The deletion removes a genomic region containing at least codons 1147 through 1854 and corresponding to the D3-A3 homologous protein domains. The extent of the vWF pseudogene on chromosome 22 is roughly similar to that of the deleted area. However, the pseudogenic nature of the deletion is excluded by the mapping of bands with reduced intensity in the patient to the true vWF gene. The vWF antigen levels are one fourth of normal and ristocetin cofactor activity is severely impaired. The reduction of high molecular weight multimers in plasma and platelets and the altered triplet morphology are compatible with the presence of a dominant variant of type II von Willebrand disease.
Acetylcholinesterase and lymphocyte function-associated antigen 3 found on decay-accelerating factor-negative erythrocytes from some patients with paroxysmal nocturnal hemoglobinuria are lost during erythrocyte aging
Authors: Ueda E , Kinoshita T , Terasawa T , Shichishima T , Yawata Y , Inoue K , Kitani T
Published: October 2019

Erythrocytes from patients with paroxysmal nocturnal hemoglobinuria are deficient in decay-accelerating factor (DAF), a factor called C8- binding protein or homologous restriction factor, acetylcholinesterase (AchE), and lymphocyte function-associated antigen 3 (LFA-3). These proteins share a common feature that glycan-inositolphospholipid anchors the protein to the membrane, suggesting that an abnormality related to this glycolipid causes multiple protein deficiencies. The relationship between the DAF, AchE, and LFA-3 defects was studied by fluorescent flow cytometric analysis. In five patients, DAF-negative erythrocytes were also AchE-negative. In three patients, a fraction of DAF-negative erythrocytes expressed subnormal levels of AchE, indicating that AchE was synthesized in these DAF-negative cells. Erythrocytes from the patients having DAF-negative, AchE-positive cells were separated according to density and analyzed for expression of DAF and AchE. Both proteins decreased with increase of cell density, suggesting that DAF-negative, AchE-positive cells become AchE-negative during erythrocyte maturation by losing AchE. A low level of LFA-3 was found on DAF-negative erythrocytes from one patient and decreased with erythrocyte maturation. These results support an idea that complete deficiency of glycan-inositolphospholipid-anchored proteins on erythrocytes could result from abnormally early termination of surface recruitment of these proteins, and subsequent dilution through cell divisions and loss from the surface.
A new hereditary persistence of fetal hemoglobin deletion has the breakpoint within the 3' beta-globin gene enhancer
Authors: Camaschella C , Serra A , Gottardi E , Alfarano A , Revello D , Mazza U , Saglio G
Published: October 2019

A new deletion of the beta-globin gene cluster has been characterized in two Italian brothers who are heterozygous carriers of a G gamma A gamma hereditary persistence of fetal hemoglobin (HPFH). Restriction endonuclease mapping and DNA sequencing of the region encompassing the breakpoint show that the deletion starts 3.2 kilobases (kb) upstream from the delta gene and ends within the enhancer region 3′ to the beta- globin gene. Here the deletion removes one of the four binding sites for an erythroid specific transcriptional factor (NF-E1). The molecular comparison of the new deletion with others of similar size and location but associated with a delta beta-thalassemia phenotype suggests that the residual enhancer element, relocated near gamma genes, may increase the fetal hemoglobin (HbF) expression above the level observed in delta beta-thalassemia.
Analysis of proto-oncogenes in acute myeloid leukemia: loss of heterozygosity for the Ha-ras gene
Authors: Ahuja HG , Foti A , Zhou DJ , Cline MJ
Published: October 2019

At least 13 of 34 patients with acute myeloid leukemia (AML) of varying FAB types were heterozygous for a BamHI restriction fragment length polymorphism (RFLP) of the Ha-ras gene on chromosome 11. In 4 of these 13 patients, one allele of the Ha-ras gene was deleted. Two of these cases had an informative heterozygosity for an RFLP on the long arm of chromosome 11. Analysis of these cases indicated that loss of genes from chromosome 11 was restricted to the short arm. In three cases with loss of one Ha-ras gene, the remaining gene had no mutations in critical areas of exons 1 and 2. With the exception of one AML case with amplification of MYC, no gross structural abnormalities in 12 other oncogenes were detected.
Identification of an abnormal gene for the GPIIIa subunit of the platelet fibrinogen receptor resulting in Glanzmann's thrombasthenia
Authors: Bray PF , Shuman MA
Published: October 2019

The platelet fibrinogen receptor, which is composed of glycoproteins IIb (GPIIb) and IIIa (GPIIIa), belongs to a large family of receptors that participate in a multitude of biologically important adhesive interactions. Platelets from most patients with the autosomal recessive bleeding disorder, Glanzmann's thrombasthenia, are deficient in GPIIb and GPIIIa. We have used cDNA probes to analyze the GPIIb and GPIIIa genes in four patients from three kindreds with Glanzmann's thrombasthenia. Southern analysis of their DNA was identical to that observed in normals when probed with a full-length GPIIb cDNA or a 3′ GPIIIa cDNA. However, in one family, a 5′ 2.0 kb GPIIIa cDNA identified abnormal DNA fragments in the father and two affected siblings' genes. A series of restriction digests resulting in small genomic fragments were probed with portions of the 5′ 2.0 kb GPIIIa cDNA and indicated that the abnormal sequences are flanked by normal fragments of the GPIIIa gene. To analyze further the genetic defect in this family, RNA was prepared from their platelets. Northern analysis revealed normal levels of GPIIb mRNA compared to control platelets. We were unable to identify GPIIIa mRNA of any size in the clinically affected family members. We also identified an EcoRI restriction fragment length polymorphism (RFLP) that permitted carrier status determination in the clinically unaffected siblings. These studies indicate that Glanzmann's thrombasthenia can be caused by heterogeneous defects in the GPIIIa gene. Furthermore, we have shown that platelets can be used to characterize normal and abnormal GPIIIa and GPIIb mRNA, and RFLPs may be used to determine the carrier status in some families with Glanzmann's thrombasthenia. The specific gene abnormality in this family appears to represent an example of an insertional mutation resulting in a human disease.
Study of the CD3-associated T-cell receptors reveals further differences between T-cell acute lymphoblastic lymphoma and leukemia
Authors: Gouttefangeas C , Bensussan A , Boumsell L
Published: October 2019

We show further differences between two clinically related entities, T- cell acute lymphoblastic leukemia (T-ALL) and lymphoblastic lymphoma (T- LL), by using several monoclonal antibodies (MoAbs) reacting with either constant or variable regions of T-cell receptors (TcR) alpha beta and gamma delta or with various CD molecules. We analyzed a panel of 15 T-ALL and 15 T-LL selected for their cell surface expression of the CD3 molecules. The results indicated that TcR gamma delta is more frequently used than TcR alpha beta in T-ALL (10 of the 15 patients tested). This is in contrast to the results obtained with T-LL where the vast majority expressed TcR alpha beta (13 of the 15 patients). These findings suggest that the leukemic cells could have a different origin in these two diseases. In addition, analysis of TcR variable regions expressed by the leukemic blasts showed that, in most cases, they had rearranged functional V delta 1 gene segments (8 of 11 patients), whereas in a unique case V delta 2 gene segment was used. Together, these results and those indicating that T-ALL cells coexpress the CD1a, b, and c molecules strengthen the possibility that although these leukemic cells express the CD3-TcR complex at their cell surface, their normal counterparts are not found in peripheral blood.
Human gamma delta T-cell receptor-positive cell-mediated inhibition of erythropoiesis in vitro in a patient with type I autoimmune polyglandular syndrome and pure red blood cell aplasia [see comments]
Authors: Hara T , Mizuno Y , Nagata M , Okabe Y , Taniguchi S , Harada M , Niho Y , Oshimi K , Ohga S , Yoshikai Y
Published: October 2019

The gamma delta T-cell receptor-positive (gamma delta TCR+) lymphocytes were markedly expanded up to 68% of peripheral blood lymphocytes in a case with type I autoimmune polyglandular syndrome and pure red blood cell aplasia (PRCA). The gamma delta TCR+ cells showed CD4 negative, 16% dim-CD8 positive and 10% to 46% human leukocyte antigen-D-related (HLA-DR) positive, and exhibited no monoclonality as assessed by the patterns of TCR gene rearrangements. Functional studies revealed that the proliferative responses of the patient's peripheral blood mononuclear cells (PBMC) were severely depressed to candida antigen, alloantigens, and autoantigens (non-T cells). The gamma delta TCR+ cells had no suppressive effect on the proliferative response of the alpha beta TCR+ cells to candida. The patient's PBMC, isolated gamma delta TCR+ cells but not alpha beta TCR+ cells, exhibited non-major histocompatibility complex (MHC)-restricted cytotoxicity. Furthermore, the patient's PBMC and isolated gamma delta TCR+ cells inhibited burst- forming units-erythroid (BFU-E), but not colony-forming units/granulocyte-macrophage (CFU-GM). Supernatants derived from the patient's T cells similarly inhibited BFU-E but not CFU-GM. The clinical course of the patient also showed a close correlation between the decreased number of total lymphocyte counts, especially HLA-DR + gamma delta TCR+ cell counts, and recovery from PRCA. These observations suggest that the gamma delta TCR+ cells might be functional in vivo and involved in the pathogenesis of PRCA in this patient.
Development, characterization, and subcellular location of DNAse activity in HL-60 cells and monocytes
Authors: Roberts PJ
Published: October 2019

The digestion of DNA from intact bacteria by human phagocytic cells was measured by the release of solubilized radiolabeled DNA. Two subclones from the human promyelocytic HL-60 cell line were unable to digest bacterial DNA unless they were previously induced to mature by incubation for several days with 1.25% dimethylsulfoxide (DMSO). The maximal capacity of DMSO-induced HL-60 cells to digest DNA was similar to that of monocytes purified from peripheral blood (PB) and much greater than that of neutrophils. The increasing capacity to digest DNA during maturation was associated with the development of acid DNAse activity, measured in a cell-free system, and slightly preceded development of 12-O-tetradecanoyl phorbol 13-acetate-stimulated respiratory burst activity. The acid DNAse had a pH optimum of 5.0 and did not require the presence of calcium or 2-mercaptoethanol (2-ME). A third subclone of HL-60 cells was able to digest DNA from intact bacteria without previous maturation, however, and this was associated with the presence of an alkaline DNAse which had a pH optimum between 7.0 and 8.0 and showed a dependence on calcium and 2-ME for maximal activity. The subcellular location of acid DNAse in DMSO-induced HL-60 cells was similar to that of monocytes in having a bimodal distribution on fractionated sucrose density gradients. The dense peak (mean density 1.195 g/mL) was located in the same region of the gradient as primary granule enzymes but the light peak (mean density 1.137 g/mL) did not codistribute with either plasma membrane, endoplasmic reticulum, or mitochondria, suggesting accumulation in a different organelle.
Induction of platelet Ca2+ influx and mobilization by a monoclonal antibody to CD9 antigen
Authors: Hato T , Sumida M , Yasukawa M , Watanabe A , Okuda H , Kobayashi Y
Published: October 2019

We found that a monoclonal antibody (MoAb) to CD9 antigen, PMA2, induced a rise in cytosolic free calcium concentration ([Ca2+]i) in fura-2-loaded platelets, and we examined whether this response was due to direct action of PMA2 on CD9 antigen. The rise in [Ca2+]i was dependent on the PMA2 concentration, irrespective of the presence or absence of extracellular Ca2+. The role of secreted adenosine diphosphate (ADP) and thromboxane in the [Ca2+]i response to PMA2 was studied using creatine phosphate/creatine phosphokinase (CP/CPK) and aspirin. Combined treatment with CP/CPK and aspirin abolished the rise in [Ca2+]i, although either CP/CPK or aspirin alone produced only partial inhibition. Inhibition of adenosine triphosphate (ATP) secretion and thromboxane B2 synthesis by an MoAb to the glycoprotein IIb-IIIa complex, PMA1, resulted in little [Ca2+]i response to PMA2. In contrast, thrombasthenic platelets, in which ATP secretion and thromboxane B2 synthesis were normal, showed a normal [Ca2+]i response. When PMA2 was added to CD9+ mononuclear cells, no rise in [Ca2+]i was observed. Thus, we conclude that binding of monoclonal immunoglobulin G molecules to the CD9 antigen raises [Ca2+]i through the effect of secreted ADP and thromboxane on platelets, and that CD9 antigen is not directly involved in induction of Ca2+ influx and mobilization.
Membrane expression of decay-accelerating factor on neutrophils from normal individuals and patients with paroxysmal nocturnal hemoglobinuria
Authors: Okuda K , Kanamaru A , Ueda E , Kitani T , Nagai K
Published: October 2019

Decay-accelerating factor (DAF), a complement-regulating glycoprotein, was found to be a maturational protein for normal neutrophils, and a remarkable correlation was found between the DAF level and alkaline phosphatase activity in neutrophils. We studied the relationship between the amount of DAF on the membrane and cell maturity in total nucleated bone marrow (BM) cells, mature BM and peripheral blood (PB) neutrophils from normal subjects, and patients with paroxysmal nocturnal hemoglobinuria (PNH) using a fluorescence-activated cell sorter with anti-DAF monoclonal antibodies. Percentage distributions of differentiating neutrophils from normal total nucleated BM cells showed that the proportion of immature cells (myeloblasts plus promyelocytes) decreased, while that of mature ones (bands plus segmented forms) increased as the fluorescence intensity increased. For PB neutrophils, no apparent correlation was found between DAF expression and cell maturity. This may have resulted from margination of the fully matured neutrophils with a high amount of DAF. In PNH patients who have low levels of DAF, this study showed that DAF expression in their neutrophils differs from that in normal subjects, and abnormalities occur in PNH cells from a very early stem-cell stage.
Biologic and biochemic properties of recombinant platelet factor 4 demonstrate identity with the native protein
Authors: Park KS , Rifat S , Eck H , Adachi K , Surrey S , Poncz M
Published: October 2019

Platelet factor 4 (PF4) is a 70 amino acid protein released from the alpha-granules of platelets after activation. The exact biologic function of this protein is unknown. We have constructed an expression vector for recombinant PF4 (rPF4) in the T7-based promoter vector pT7–7 to better study the relationship between PF4 structure and function. The protein was expressed in Escherichia coli and purified to homogeneity by heparin-agarose affinity chromatography and reverse- phase high-performance liquid chromatography. Purity of protein was confirmed by immunoblot analysis and sodium dodecyl sulfate- polyacrylamide gel electrophoresis, which resulted in a single component with a molecular weight of 8,000 daltons. The amino acid composition and sequence of the N-terminal 20 residues showed that rPF4 is identical to PF4 prepared from human platelets (hPF4), except for an N-terminal initiating methionine residue. Immunoblots revealed that rPF4 and hPF4 bound polyclonal anti-hPF4 equally well, while chemotaxis experiments demonstrated similar potencies as neutrophil attractants. Dose-dependent neutrophil chemotactic responses and competitive studies with polyclonal anti-hPF4 antiserum further demonstrate similar chemotactic properties of the two PF4 species. In conclusion, our data show that this recombinant protein and the native protein appears to have similar immunologic, heparin-binding, and chemotactic properties. The chemotactic properties of hPF4 appear to be entirely intrinsic to the protein and not due, in part, to any contaminating protein. Furthermore, our expression vector should prove useful for the construction of recombinant forms of PF4 to investigate structure/function relationships of this biologically important protein.
Use of an anti-pan T-lymphocyte ricin a chain immunotoxin in steroid- resistant acute graft-versus-host disease
Authors: Byers VS , Henslee PJ , Kernan NA , Blazar BR , Gingrich R , Phillips GL , LeMaistre CF , Gilliland G , Antin JH , Martin P
Published: October 2019

Acute steroid-resistant graft-versus-host disease (AGVHD) after allogeneic bone marrow transplantation is frequently fatal. A new treatment for this T-lymphocyte-mediated condition uses an immunotoxin, H65-RTA, comprised of a monoclonal antibody that recognizes the CD5 lymphocyte differentiation antigen coupled to ricin A chain, a cytotoxic enzyme that inhibits protein synthesis. The safety and efficacy of this lymphocyte-targeted immunotoxin was evaluated in patients with severe AGVHD in a phase I-II dose escalation study with group expansion at the two middle doses. Thirty-four patients received up to 14 daily intravenous infusions of the immunotoxin. The principal side effects were constitutional symptoms such as fatigue and myalgias, and hypoalbuminemia with weight gain was seen at all doses. Thirty-two patients were evaluated for improvement or resolution of disease. Durable complete or partial responses were not dose-related and were seen in 16 patients. Skin GVHD had the highest incidence of response (73%), although improvement or resolution in gastrointestinal tract (45%) and liver (28%) GVHD was also noted. Survival in responding patients was significantly prolonged at all times as compared with those with no response (P = .03). Treatment was associated with a rapid decrease in peripheral blood T lymphocytes, which persisted for greater than 1 month after therapy. Anti-immunotoxin antibodies were seen in 6 of the 23 patients tested; these were of low titer and did not block immunotoxin binding to T cells. Results of this study indicate that anti-T-lymphocyte immunotoxins may form a new class of immunosuppressive agents useful in T-lymphocyte-mediated diseases.
Common binding structure for granulocyte macrophage colony-stimulating factor and interleukin-3 on human acute myeloid leukemia cells and monocytes
Authors: Budel LM , Elbaz O , Hoogerbrugge H , Delwel R , Mahmoud LA , Lowenberg B , Touw IP
Published: October 2019

Granulocyte macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3) control the proliferation of human acute myeloid leukemia (AML) cells in vitro. Previously, we have shown that receptors for GM-CSF and IL-3 are often coexpressed on AML cells. Here we present experiments with purified AML blasts, normal monocytes, and granulocytes that were conducted to analyze the properties of GM-CSF and IL-3 binding proteins in more detail. On AML cells from eight cases we demonstrate two types of GM-CSF receptors: one with low affinity (dissociation constant [kd] 5.1 to 24.8 nmol/L) and one with a high affinity (kd 31 to 104 pmol/L). These AML cells also expressed high affinity receptors for IL-3 (kd 24 to 104 pmol/L). Cross-competition experiments showed that an excess concentration of nonlabeled IL-3 completely prevented the high affinity binding of radiolabled GM-CSF. This competition occurred at 37 degrees C as well as 4 degrees C. Low affinity GM-CSF binding was not affected by IL-3. Binding of radiolabeled IL-3 could be prevented by nonlabeled GM-CSF. In certain cases, this competition was complete, whereas in others only partial (49% to 77%) reduction of the radiolabeled IL-3 binding was seen. On the basis of these ligand binding features, we propose the existence of three receptor types on AML cells: (1) low affinity GM-CSF receptors that do not bind IL-3, (2) dual high affinity GM-CSF/IL-3 receptors, and (3) high affinity IL-3 receptors that do not bind GM-CSF. We could also demonstrate these receptor types on normal monocytes. In addition, a fourth type of receptor was apparent on normal granulocytes (4), incapable of binding IL-3 and with an intermediate affinity for GM-CSF (approximately 400 pmol/L). Chemical crosslinking showed that GM-CSF and IL-3 both bind to proteins with molecular weight values of 130, 105, and 75, which provides additional evidence for the existence of a common GM-CSF/IL-3 receptor complex.
Characterization and fibrinolytic properties of human omental tissue mesothelial cells. Comparison with endothelial cells
Authors: van Hinsbergh VW , Kooistra T , Scheffer MA , Hajo van Bockel J , van Muijen GN
Published: October 2019

It has been reported that omental fat tissue is a good source of human microvascular endothelial cells. By characterization we demonstrate that the epitheloid cells isolated from omental tissue are not endothelial cells, but mesothelial cells. They contain abundant cytokeratins 8 and 18, which are absent in endothelial cells, and vimentin. No staining with the endothelial-specific antibodies EN-4 and PAL-E is observed. A faint and diffuse staining of von Willebrand factor (vWF) is seen in mesothelial cells, whereas microvascular endothelial cells from subcutaneous fat display vWF in distinct granular structures. Human peritoneal mesothelium produces plasminogen activator-dependent fibrinolytic activity, which is essential in the resolution of fibrous exudates and may therefore be important in preventing the formation of fibrous peritoneal adhesions. This fibrinolytic activity is plasminogen activator-dependent, but has not been fully characterized. We report here that human omental tissue mesothelial cells in vitro produce large amounts of tissue-type plasminogen activator (t-PA), together with type 1 and 2 plasminogen activator inhibitor (PAI-1 and PAI-2). PAI-1 is predominantly secreted into the culture medium, whereas the major part of PAI-2 is found in the cells. No urokinase-type plasminogen activator is detected. On stimulation with the inflammatory mediator tumor necrosis factor (TNF), at least a threefold decrease in t-PA antigen is observed, together with an increase in both PAI-1 and PAI-2. TNF also induces a marked change in cell shape. Whereas TNF and bacterial lipopolysaccharide (LPS) have similar effects on the production of PA inhibitor by human endothelial cells, LPS has no or only a relatively small effect on the fibrinolytic properties of mesothelial cells. The decreased fibrinolytic activity induced by the cytokine TNF may impair the natural dissolution of fibrin deposits at the peritoneum in the presence of an inflammatory reaction.
Characterization of a new T-lineage glycoprotein expressed in mature T- cell leukemias and lymphomas
Authors: Cassano WF
Published: October 2019

We identified a new human, T-lineage restricted glycoprotein of molecular weight 120 Kd that is expressed primarily in mature T-cell malignancies. The antigen, named TCA-1 (T-cell cytoplasmic antigen), is an intracellular glycoprotein found mainly in the Golgi stacks, although a few cell lines also display surface membrane TCA-1. Many but not all T-cell neoplasms express this antigen. The antigen is absent from neoplastic and normal human tissue outside the T-lymphocyte lineage. TCA-1 was identified by murine monoclonal antibodies produced after immunization of mice with T-cell chronic lymphocytic leukemia cells. The glycoprotein is a monomer containing approximately 4% N- linked carbohydrate with terminal D-galactose residues. Partial amino acid sequence analysis of TCA-1 shows homology with an immunoglobulin heavy chain region, which suggests that TCA-1 may belong to the immunoglobulin supergene family of receptor and adhesion molecules.
Dynamics of erythropoietin receptor expression on erythropoietin- responsive murine cell lines
Authors: Broudy VC , Nakamoto B , Lin N , Papayannopoulou T
Published: October 2019

We examined erythropoietin receptor expression in two murine cell lines, B6SUtA and DA-1, that respond to erythropoietin in different ways. While B6SUtA cells undergo erythroid differentiation with limited proliferation after addition of erythropoietin, DA-1 cells show only a proliferative response. Equilibrium binding experiments with 125I- erythropoietin revealed that both B6SUtA and DA-1 cells express a single class of erythropoietin receptors. In the absence of erythropoietin, B6SUtA cells exhibited 145 receptors per cell with a dissociation constant (kd) of 380 pmol/L. Six days after induction with erythropoietin, the B6SUtA cells displayed 310 receptors per cell without a change in binding affinity; exposure to erythropoietin also increased cellular hemoglobin content. DA-1 cells adapted to erythropoietin-dependent growth over a period of months and exhibited a progressive increase in erythropoietin receptor expression, from 85 per cell (kd = 540) to 550 per cell (kd = 530), although the cells remained uniformly benzidine-negative. We interpret the data with B6SUtA cells to indicate that early erythroid differentiation stages are attended by an increase in erythropoietin receptor display, coordinate with the initiation of expression of erythroid-specific genes. In contrast, the results with DA-1 cells are most compatible with clonal selection as the mechanism underlying enhanced receptor expression. Thus, display of the erythropoietin receptor is dynamic and can be modulated during the course of erythropoietin-induced differentiation.
Phorbol 12-myristate 13-acetate-induced development of functionally active mast cells in W/Wv but not Sl/Sld genetically mast cell- deficient mice
Authors: Gordon JR , Galli SJ
Published: October 2019

The normal skin and other tissues of adult genetically mast cell- deficient WBB6F1-W/Wv or WCB6F1-Sl/Sld mice contain less than 1.0% the number of mast cells present in the corresponding tissues of the congenic normal (+/+) mice. We previously reported that mature dermal mast cells developed locally in the skin of W/Wv, but not Sl/Sld, mice at sites of chronic idiopathic dermatitis. We now report that the repeated application of phorbol 12-myristate 13-acetate (PMA) to the ear skin of either W/Wv or +/+ mice induces both dermatitis and a striking and dose-dependent increase in the number of dermal mast cells. The number of dermal mast cells at sites treated for 6 weeks with 5 micrograms PMA, three times per week, was 39 +/- 7/mm2 and 305 +/- 34/mm2 for W/Wv and +/+ mice, respectively; the corresponding values for vehicle-treated skin were 1.5 +/- 1.0/mm2 and 145 +/- 8/mm2, respectively. The PMA-induced dermal mast cells in W/Wv mice appeared mature by morphology, stained with the heparin-binding fluorescent dye, berberine sulfate, and were competent to express IgE-dependent passive cutaneous anaphylaxis responses. The development of mast cells was a local, not systemic, effect of PMA treatment. PMA treatment also induced dermatitis in both WCB6F1-Sl/Sld and +/+ mice, but was associated with increased numbers of dermal mast cells only in the WCB6F1(-)+/+ mice. PMA treatment had no detectable effect on the ability of bone marrow-derived cultured mast cells to survive in the skin of Sl/Sld mice. These findings establish a convenient model system for analyzing factors associated with the development of endogenous populations of mast cells in genetically mast cell-deficient W/Wv mice.
Human colony-stimulating factor 1 (CSF-1) receptor confers CSF-1 responsiveness to interleukin-3-dependent 32DC13 mouse myeloid cells and abrogates differentiation in response to granulocyte CSF
Authors: Kato J , Sherr CJ
Published: October 2019

Interleukin-3 (IL-3)-dependent mouse myeloid 32DC13 cells differentiate to neutrophils in response to granulocyte colony-stimulating factor (G- CSF). Introduction of the human c-fms gene, which encodes the receptor for CSF-1, into 32DC13 cells gave rise to variants that were able to proliferate in medium containing either murine IL-3 or human recombinant CSF-1, but were unable to differentiate to granulocytes in response to G-CSF. Unlike parental 32CD13 cells, CSF-1-responsive derivatives expressed nonspecific esterase when grown in CSF-1, but did not exhibit many other morphologic, immunologic, or functional properties of mononuclear phagocyte differentiation, or express murine CSF-1 receptors. Accelerated turnover of the human CSF-1 receptor was observed in response to CSF-1 and phorbol esters, but not after stimulation with IL-3 or bacterial lipopolysaccharide. Although both CSF-1 and IL-3 induced tyrosine phosphorylation of heterologous substrates in the dually responsive cells, differences in the patterns of substrate phosphorylation were observed in response to the two hematopoietins. We conclude that expression of the human CSF-1 receptor in 32DC13 cells not only induces CSF-1 responsiveness, but alters its phenotype in a way that prohibits granulocyte differentiation.
Analyses of thrombocytopenia in idiopathic thrombocytopenic purpura- prone mice by platelet transfer experiments between (NZW x BXSB)F1 and normal mice
Authors: Mizutani H , Furubayashi T , Kuriu A , Take H , Tomiyama Y , Yoshida H , Nakamura Y , Inaba M , Kurata Y , Yonezawa T
Published: October 2019

Male (NZW x BXSB) F1 (W/B F1) mice, which develop lupus nephritis, myocardial infarction, and thrombocytopenia, showed reduced platelet lifespan (PLS) and increased platelet-associated antibody (PAA) values. There were statistically significant correlations between the increase in PAA values and either the reduction in PLS or the decrease in platelet counts. This and the results of platelet transfer experiments between old male W/B F1 mice and either female W/B F1 or normal BALB/c mice indicate that PAAs on the platelet surface play a crucial role in the destruction of platelets in W/B F1 mice. The mechanism of thrombocytopenia observed here appears similar to that of human idiopathic thrombocytopenic purpura (ITP). Therefore, we think that W/B F1 mice are a potentially useful animal model for investigating the effectiveness and mode of action of therapeutic agents in human ITP, and that they may provide additional information on the basic mechanisms of this autoimmune phenomenon.