Monoclonal antibody J11d.2 recognizes cell cycle-dormant, primitive hematopoietic progenitors of mice

It was reported that monoclonal antibody (MoAb) J11d.2 reacts with mature blood cells of mice but not with their progenitors. We tested in culture studies whether this antibody could be used for enrichment for primitive marrow progenitors. The majority of colony-forming cells including multipotential progenitors in the marrow cells from 5- fluorouracil (5-FU)-treated mice were J11d.2+, whereas most of the progenitors from normal mice were J11d.2-. In addition, formation of multilineage colonies from J11d.2+ in both 5-FU-treated and normal mice was augmented by interleukin 6. These observations indicated that MoAb J11d.2 recognizes cell cycle-dormant progenitors. We have recently described a simple method that provides 800-fold enrichment for the progenitors in post-5-FU marrow cells using MoAb D7 (anti-Ly-6A/E). When this method was modified to include sorting with MoAb J11d.2, D7+ J11d.2+ cells were 2,250-fold enriched for multipotential progenitors. Micromanipulation and culture of individual D7+ J11d.2+ cells showed that average plating efficiency of the cell population is approximately 70% and that about 30% of the progenitors are lymphohematopoietic in nature. These data demonstrate that J11d.2 is a useful MoAb for the isolation of primitive hematopoietic progenitors of mice.     

New variant of von Willebrand disease with defective binding to factor VIII

A new variant of von Willebrand disease (vWD) was identified by a new analytic method which characterizes the ability of plasma von Willebrand Factor (vWF) to bind to purified factor VIII (F.VIII). vWF was isolated from small amounts of plasma by immunoadsorption with a selected monoclonal antibody to vWF previously coated onto wells of microtitration plates. Plasma F.VIII was removed from immobilized vWF by washing with 0.4 mol/L CaCl2; purified F.VIII was then added to the well. The amount of bound F.VIII was estimated directly in the wells by a chromogenic assay and immobilized vWF was estimated by an immunologic a pool of normal plasma, ten control individuals, 13 with hemophilia A and five with type I vWD. In all cases, the dose-response curves were linear and the slopes of the regression lines were essentially the same. The method was then applied to investigate the binding of vWF to F.VIII in two vWD patients (sister and brother) who demonstrated significantly lower activity of F.VIII than of vWF. The first patient, with a long history of epistaxis, bruising, and hematomas, showed a slightly prolonged bleeding time (10 minutes); 15% VIII:C and 39% of vWF:Ag and vWFRCo. Her brother, who has a bleeding syndrome but no hematomas, showed similar data (bleeding time 9 minutes, 20% VIII:C, 53% vWF:Ag and vWFRCo). Similar levels of F.VIII were observed in the two propositi by four different methods (one- and two-stage clotting and chromogenic and immunologic assays). Sodium dodecyl sulfate (SDS) 1.4% agarose gel electrophoresis showed that all multimers of vWF were present in both patients. vWF binding to F.VIII was markedly decreased in the two propositi. The abnormal binding of vWF to F.VIII was not corrected during pregnancy or after infusion of 1-deamino (8-D- arginine) vasopressin despite an increase in vWF levels. The qualitative abnormality of vWF in both patients was associated with a subtle alteration of the multimeric structure by SDS 3% agarose gel electrophoresis in which the two central subbands of the quintuplet of individual oligomers were undetectable or poorly visible. SDS- polyacrylamide gel electrophoresis under reducing conditions demonstrated a single band of 275 Kd in the plasma of both patients, and there was no evidence of a second band corresponding to pro-vWF, the precursor of the mature vWF subunit, suggesting that proteolytic processing of vWF was normal.(ABSTRACT TRUNCATED AT 400 WORDS)     

A pilot study of continuous ambulatory electrocardiography in patients donating blood for autologous use in elective coronary artery bypass grafting

BACKGROUND: A pilot study was conducted to evaluate the impact of a single autologous blood donation on the presence or absence of myocardial ischemic episodes in patients with coronary artery disease. STUDY DESIGN AND METHODS: Fifty patients scheduled for elective coronary artery bypass grafting underwent two 24-hour periods of ambulatory electrocardiogram monitoring, one before and one after their first autologous blood donation. The presence or absence and the number, duration, and integral area of episodes of ST segment depression for each 24-hour monitoring period were determined. RESULTS: Forty-two patients had legible electrocardiogram recordings for both monitoring periods. Of these, 36 patients (86%) had at least one episode of ST segment depression during any monitoring period. The number of patients who had at least one episode of ST segment depression before donation was not significantly different from the number of those who had at least one episode after donation (31 and 33 patients, respectively; p = 0.73). CONCLUSION: Donating a unit of blood had no demonstrable effect on the presence or absence of myocardial ischemic episodes in this sample of 42 autologous blood donors with coronary artery disease. The results of this study should be validated in further trials.     

Opposite effects of tumor necrosis factor alpha on the sphingomyelin- ceramide pathway in two myeloid leukemia cell lines: role of transverse sphingomyelin distribution in the plasma membrane

Tumor necrosis factor alpha (TNF alpha) mediates proliferation, functional activation, and apoptotic cell death depending on the target cell type. Although sphingomyelin (SPM) hydrolysis and ceramide generation may function as an important mediator in TNF alpha signaling, the molecular mechanisms of the signaling pathway(s) are still not well understood. The aim of the present study is to compare the effect of TNF alpha on SPM metabolism and cell growth in two myeloid leukemic cell lines (U937 and KG1a) that differ in their sensitivity to TNF alpha. Our results show that TNF alpha induced apoptosis in U937 but not in KG1a cells. TNF alpha triggered in KG1a cells neither SPM hydrolysis nor ceramide generation, but induced SPM synthesis and ceramide breakdown as well as dose-dependent cell proliferation. In contrast, TNF alpha induced in U937 SPM hydrolysis and ceramide generation as well as dose-dependent cell death. Synthetic cell permeant ceramide, as well as natural ceramide, generated by treatment with bacterial sphingomyelinase (SPMase), all induced apoptosis in both U937 and KG1a cells. These findings indicate that the SPM-ceramide pathway is altered in KG1a cells upstream of the ceramide generation. Analysis of the transverse distribution of SPM in the plasma membrane showed that the SPM pool involved in cell signaling (inner leaflet) was markedly reduced in KG1a cells; it is 7-fold lower than that found in the inner leaflet of U937 cells. Therefore, our study strongly suggests that the different responses induced by TNF alpha in myeloid cells are dependent on the SPM plasma membrane transverse asymmetry.     

Clinical factors influencing the efficacy of pooled platelet transfusions

To determine the relative importance of clinical factors on the efficacy of platelet transfusions, 941 pooled platelet transfusions from HLA-unmatched donors were studied prospectively in 133 patients with bone marrow failure. Multiple linear regression analyses identified the major factors influencing one-hour-corrected increments (CI) as prior splenectomy, bone marrow transplantation, disseminated intravascular coagulation, concurrent intravenous amphotericin B, splenomegaly, and HLA antibody grade. The relative impact of these factors on CI has been quantitated by using a formula developed from these data. A linear relationship was demonstrated between increasing percentage of HLA antibody grade and decreasing CI. A number of other factors were less important in the linear regression model than the aforementioned major factors. These included platelet-specific antibodies, concurrent antibacterial antibiotics, clinical bleeding grade, and temperature. Factors that did not influence CI included the number of prior platelet transfusions, prior granulocyte transfusions, prior red cell transfusions, infection, age, blood group, diagnosis, sex, pretransfusion platelet count, prior pregnancies, and concurrent antineoplastic drugs. This study identified major clinical factors that significantly influenced CI and were major causes of refractoriness to pooled platelet transfusions.     

KRDS--a tetrapeptide derived from lactotransferrin--inhibits binding of monoclonal antibody against glycoprotein IIb-IIIa on ADP-stimulated platelets and megakaryocytes

Short peptides isolated from fibrinogen and K-casein have been shown to inhibit platelet aggregation and fibrinogen binding to stimulated platelets. We studied the effects of synthetic peptides occurring in milk proteins (bovine K-casein, KNQDK, and human lactotransferrin, KRDS) and in fibrinogen (RGDS and L10) on subsequent binding of monoclonal antibodies (MoAb) against the glycoprotein (GP) IIb-IIIa complex (AP2 and P2) on adenosine diphosphate (ADP)-stimulated and unstimulated human platelets and megakaryocytes (MKs) by using an immunoperoxidase method to visualize antibody binding. Only KRDS (900 mumol/L) inhibited the binding of AP2 and P2 on ADP (5 mumol/L)- stimulated platelets, but not on unstimulated platelets. However, the binding of P2 was considerably more inhibited than that of AP2 as judged by immunoperoxidase intensity. Radiolabeled AP2 binding was inhibited by 30% with KRDS on ADP-stimulated platelets as compared with platelets incubated in the absence of ADP. KRDS did not inhibit the binding of MoAbs against GP IIIa (SZ 21), GP IIb (SZ 22), and GP Ib (SZ 2) on ADP-stimulated human platelets. Inhibition of P2 binding by KRDS was also observed in a section of MKs isolated from human bone marrow and stimulated by 15 or 20 micron ADP. A lower concentration of ADP (5 or 10 mumol/L) failed to produce any inhibition of binding. This indicates that MKs may not be equally responsive to agonists as platelets. Moreover, P2 binding inhibition was observed in a larger (P less than .001) percentage of mature MKs (29%) as compared with younger, maturing MKs (11%). The observations suggested that a functional ability possessed by platelets, namely, agonist-induced exposure of the site of interaction of KRDS, may occur at a late stage of MK development.     

Anatomy of the acromial arch: correlation of anatomy and magnetic resonance imaging

Summary Based on a retrospective study of 179 MRI records covering four populations (patients presenting with impingement without known injury (n=90), post-traumatic shoulder pain (n=28), instability or dislocation (n=36) and controls (n=25)), morphologic criteria are suggested to define presumedly normal arches and arches compatible with subacromial impingement. The subacromial arch is presumed normal or without impingement if the sagittal and frontal views show it to be parallel to the humeral head, and/or if there is a fatty layer interposed between the arch and the supraspinatus m. The arch is presumed aggressive or actually capable of giving rise to impingement if, in either the sagittal or frontal view, there is a zone of narrowing of the subacromial passage with an impression of the arch on the supraspinatus tendon or tendinous thinning at this level or just lateral to this narrowed zone. Based on these criteria, study of the 179 MRI records demonstrated a significant difference of distribution of the arches in the four populations. Aggressive arches were found in 45.5% of patients with impingement, 25% of patients with posttraumatic pain, 8.9% of patients with an acute or recurrent dislocation and 12% of controls. Conversely, a presumedly normal arch was found in 56% of the controls, 55% of patients with dislocation, 25% of posttraumatic painful shoulders and only 5.5% of patients with clinical impingement. Subacromial impingement may be due to the type 3 acromial dysplasia described by Bigliani or to a thickening of the coracoacromial ligament at its acromial attachment. This study was supplemented by 15 anatomic dissections which confirmed the regularity of attachment of the coracoacromial ligament at the inferior aspect of the acromion along its lateral border.

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Anatomie de la voûte acromiale : corrélation entre anatomie et imagerie par résonance magnétique

Résumé A partir de la lecture rétrospective de 179 dossiers d'IRM représentant quatre populations (patients présentant un conflit sans traumatisme reconnu (n=90), une douleur d'épaule post-traumatique (n=28), une instabilité ou une luxation (n=36) et sujets témoins (n=25)), les auteurs proposent des critères morphologiques pour définir des voûtes présumées normales et des voûtes susceptibles de rendre compte d'un conflit sous acromial. La voûte sous acromiale est présumée normale ou non conflictuelle si, sur les vues sagittales et frontales, elle apparait parallèle à la tête humérale et/ou il existe un liseré graisseux qui s'interpose entre la voûte et le m. supra-épineux. La voûte sous acromiale est présumée agressive ou susceptible de rendre compte d'un conflit si, sur l'une des vues sagittale ou frontale, il est observé une zone de rétrécissement du défilé sous acromial avec une empreinte de la voûte sur le tendon du m. supra-épineux ou un amincissement tendineux au niveau ou juste en dehors de cette zone de rétrécissement. A partir de ces critères, la lecture des 179 dossiers d'IRM permet de mettre en évidence une différence significative de répartition des voûtes dans les quatre populations. Les voûtes agressives s'observent chez 45.5 % des patients présentant un conflit, 25 % des patients pr%esentant une douleur au décours d'un traumatisme, 8.9 % des sujets examinés au décours d'une luxation aigue ou récidivante et 12 % des sujets témoins. A l'inverse, la voûte présumée normale est retrouvée chez 56 % des sujets témoins, 55 % des patients examinés pour une luxation, 25 % des douleurs d'épaule post traumatique et seulement 5.5 % des patients présentant un conflit clinique. Le conflit sous acromial peut être le fait d'une dysplasie acromiale de type 3 décrit par Bigliani ou d'un épaississement du ligament au niveau de son insertion acromiale. Ce travail est complété par 15 dissections anatomiques qui confirment la constance de l'insertion du ligament coraco-acromial sur la face inférieure de l'acromion le long de son bord latéral.

     

Binding of G-CSF, GM-CSF, tumor necrosis factor-alpha, and gamma- interferon to cell surface receptors on human myeloid leukemia cells triggers rapid tyrosine and serine phosphorylation of a 75-Kd protein

Granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), gamma-interferon (gamma-IFN), or tumor necrosis factor-alpha (TNF-alpha) triggered the rapid, stable phosphorylation of a 75-Kd protein (p75) when incubated with permeabilized HL60 human myeloid leukemia cells in the presence of [gamma-32P] ATP. Among several chemical inducers of HL60 cell differentiation, dimethyl sulfoxide also triggered p75 labeling, but retinoic acid or 12-O-tetradecanoylphorbol-13-acetate did not elicit this response. Pretreatment of cells with G-CSF or GM-CSF for more than 30 seconds before permeabilization rendered the p75 labeling undetectable, suggesting that ligand-stimulated labeling was rapidly completed within this time in intact cells. Phosphorylation of p75 occurred on serine and tyrosine residues. This conclusion was confirmed by direct phosphoamino acid analysis. Immunoblot analysis of lysates of intact HL60 cells that had been incubated with G-CSF, GM-CSF, IFN, or TNF confirmed that tyrosine phosphorylation of a p75 also occurred in response to these cytokines in intact cells. Pretreatment of intact HL60 cells with one biologic agent or dimethyl sulfoxide abolished p75 labeling in response to incubation of permeabilized cells with a second agent, strongly suggesting that the same protein was phosphorylated in response to these treatments. p75 labeling was strictly dependent on expression of the appropriate ligand receptor. Data suggest that activation of a tyrosine kinase system is an early response to the binding of G-CSF, GM-CSF, TNF, or IFN to their respective cell surface receptors, or to the addition of dimethyl sulfoxide, and that the resulting phosphorylation event(s) may play a role in securing common elements in the biologic responses to these agents.     

Platelet von Willebrand's antigen II: active release by aggregating agents and a marker of platelet release reaction in vivo

von Willebrand's antigen II (vW AgII), a plasma and platelet antigen immunologically and biochemically distinct from factor-VIII-related antigen (VIIIR:Ag), is decreased in von Willebrand's disease. vW AgII is released from platelets during aggregation and clotting. The release of platelet vW AgII was studied in washed platelets following aggregation by thrombin, collagen, and adenosine diphosphate (ADP). Total platelet vW AgII was 3.39 U/10(9) platelets. Thrombin and collagen yielded release of 85% and 86% of platelet vW AgII, respectively, at the highest concentrations. Release of platelet vW AgII was correlated with the release of 5-hydroxytryptamine (5HT). Release of vW AgII by collagen and thrombin was inhibited by metabolic inhibitors. In addition, collagen release of vW AgII was inhibited by aspirin. In clinical syndromes associated with intravascular platelet destruction, marked elevations of plasma vW AgII were noted. Thus, vW AgII is released by a metabolically active process from platelets during aggregation. In addition, vW AgII appears to be a marker of intravascular platelet destruction.     

Cerebrospinal fluid-iophendylate contrast on gradient-echo MR images

The effect on the signal intensities of cerebrospinal fluid (CSF) and iophendylate (Pantopaque) and on CSF-iophendylate contrast was studied in vitro with a small-nutation-angle (alpha) gradient refocused magnetic resonance (MR) imaging technique (GRASS) as alpha, repetition time (TR), and echo time (TE) were varied. CSF signal intensity was consistently greater than that of iophendylate. Therefore, retained intraspinal iophendylate may be considered in the differential diagnosis of focal areas of low signal intensity at the periphery of the spinal canal on GRASS images. At constant TE and TR, an increase in alpha from 6 degrees to 45 degrees increased the signal intensities of CSF and iophendylate but decreased CSF-iophendylate contrast. At constant alpha and TR, an increase in TE from 13 to 28 msec decreased the signal intensities of CSF and iophendylate but increased contrast. At constant alpha and TE, an increase in TR from 50 to 400 msec increased the signal intensities of CSF and iophendylate, as well as contrast. Clinical examples of the contrast behavior of retained intraspinal iophendylate on both spin-echo and GRASS images corroborate the experimental findings. Retained intraspinal iophendylate may mimic the appearance of intra-or extra-dural lesions, magnetic susceptibility artifact, and flow on gradient-echo MR images of the spine.