Philadelphia chromosome (Ph1)-negative chronic myelogenous leukemia (CML): a clonal disease with origin in a multipotent stem cell

It has been shown with glucose 6-phosphate dehydrogenase (G-6-PD) mosaicism that Ph1-positive chronic myelogenous leukemia (CML) is a clonal disease that involves multipotent hematopoietic stem cells. We now report G-6-PD studies of a 79-yr-old woman with Ph1-negative CML. Equal amounts of B and A-type activities were found in nonhematopoietic tissues, indicating that the patient was heterozygous for G-6-PD. In contrast, only A-type G-6-PD was found in marrow cells, blood erythrocytes, leukocytes, and platelets and in granulocyte-monocyte and eosinophil colonies grown from blood mononuclear cells. Unlike most cases of PH1-positive CML, colony growth in this patient increased during blastic transformation and the colonies contained only immature monocytic cells. The data indicate that in this patient, Ph1-negative CML is similar to the Ph1-positive form of the disease in involvement of multipotent stem cells and probable clonal origin, but the two disorders differ in the rapidity with which they enter blastic transformation and in the pattern of granulocyte-monocyte colony growth at that time.     

Unicellular or multicellular origin of human granulocyte-macrophage colonies in vitro

The assumption that human granulocyte-macrophage colonies have a unicellular origin and thus are true clones has been directly tested. Cells from seven females heterozygous for the common glucose-6- phosphate dehydrogenase (G-6-PD) gene (GdB) and the variant GdA were cultured in semisolid medium for granulocyte-macrophage colony growth and the enzyme type of individual colonies was determined. When the colony density was less than 20/dish, more than 95% of colonies had either type A or type B G-6-PD, but not both. At colony densities greater than 30/dish, between 15% and 75% of colonies had both enzyme types and therefore arose from more than one cell. These results are consistent with a unicellular origin for the colonies only when they are cultured at low densities. With increasing colony density, there was a greater frequency of colonies with both type A and type B activity, suggesting that accurate enumeration of committed stem cells can only be performed at low colony concentrations.     

Polymorphic glycoprotein-1 on mouse platelets: possible role of Pgp-1 and LFA-1 in antibody-dependent platelet cytotoxicity involving complement

The presence of the Pgp-1 glycoprotein on mouse platelets is demonstrated by antibody-binding techniques, by immunoprecipitation, and by transblotting using the monoclonal antibody (MoAb) C71/26 against Pgp-1. C71/26 immunoprecipitates as a broad band of mol wt 87,000 to 100,000 as determined by radioiodination of the platelet cell surface and by the 3H-sodium borohydride labeling technique. Immunoblotting showed Pgp-1 expression on platelets to be quantitatively similar to its presence on macrophages and resolved platelet Pgp-1 into two bands of mol wt 87,000 and 97,000 whereas Pgp-1 on parasite-elicited peritoneal macrophages showed 82,000 and 87,000 mol wt species. Platelets and monocyte/macrophage cells from either peripheral blood or from the peritoneal cavity showed homogeneous binding of Pgp-1 antibody to greater than 97% of cells by flow cytometry. In contrast, lymphocytes from peripheral blood or from the spleen showed a heterogeneous binding pattern with 20% to 30% of cells being negative, and the majority weakly positive. In functional studies, MoAbs against CR1 and CR3 substantially inhibited platelet immune adherence, whereas C71/26 showed only marginal inhibitor. In contrast, C71/26 and other MoAbs against Pgp-1 inhibited platelet- dependent cytotoxicity of antibody-coated sheep erythrocytes in the presence of C5-deficient mouse plasma whereas M1/70 against CR3 showed no effect. In this assay, MoAbs against the alpha- and beta-subunits of leukocyte functional molecule LFA-1 also inhibited platelet cytotoxicity. These results show that the platelet cell surface moieties Pgp-1 and LFA-1 are involved in or closely associated with antibody-dependent cellular cytotoxicity by platelets.     

Expression of a homologously recombined erythopoietin-SV40 T antigen fusion gene in mouse liver: evidence for erythropoietin production by Ito cells

We have obtained transgenic mice in which an erythropoietin-SV40 virus T antigen fusion gene is homologously recombined into the native Epo locus. This gene is expressed in a tissue-specific manner closely resembling that of the native Epo gene. Immunohistochemical detection of SV40 T antigen has been used to characterize the hepatic cell populations expressing the transgene. In mice stimulated by anaemia or hypobaric hypoxia, SV40 T antigen was demonstrated in two liver cell populations: a subset of hepatocytes and a nonparenchymal cell type. Immunohistochemical and ultrastructural characterization of these cells by light and electron microscopy showed the nonparenchymal cell type to be the Ito cells, which lie in a persinusoidal position within the space of Disse. We therefore conclude that Ito cells are the nonhepatocytic source of liver Epo production. These cells show many similarities to the Epo-producing fibroblastoid interstitial cells of the kidney.     

Kringle glycosylation in a modified human tissue plasminogen activator improves functional properties

Native tissue plasminogen activator (ntPA) has a variable glycosylation site on its kringle-2 domain. We have examined the effects of kringle glycosylation on functional properties by studying the simplified tPA molecule, tPA-6. tPA-6 is composed of kringle-2 and the serine protease domains and, like ntPA, cells expressing tPA-6 process it into two glycoforms: the monoglycosylated tPA-6-primary (tPA-6P, type II) with N- linked glycosylation at Asn-448 in the serine protease domain and diglycosylated tPA-6-variant (tPA-6V, type I) with glycosylation at Asn- 448 and at Asn-184 in kringle-2. When the two glycoforms were separated, we found that purified tPA-6V had reduced fibrin-stimulated plasminogenolytic activity toward Glu-plasminogen when compared to purified tPA-6P. However, in the presence of fibrin, tPA-6V unexpectedly exhibited a sixfold increase in selectivity toward Lys- plasminogen. In addition, tPA-6V was less susceptible than tPA-6P to plasmin-mediated conversion to the two-chain form. By site-directed mutagenesis of tPA-6, we eliminated variable glycosylation at Asn-184 and engineered a new glycosylation signal at a remnant site in the kringle. This derivative, designated tPA-6D, was secreted with complete kringle glycosylation. Like the naturally occurring tPA-6V, tPA-6D had lower rates of fibrin-stimulated Glu-plasminogen activation, increased specificity toward Lys-plasminogen, and greater resistance to plasmin digestion. Although the activity of tPA-6D could be stimulated by fibrin, its activity was not stimulated significantly by fibrinogen, and in human plasma the rate of fibrinogen depletion was reduced threefold. Although fibrin binding to kringle-2 of tPA-6D was slightly improved, there was a substantial increase in the dissociation constant (kd) for lysine binding, demonstrating a lack of correlation between these ligand-binding sites. Overall, our data demonstrate the marked effect of kringle glycosylation on functional properties. In addition, we have generated a derivative with properties that potentially improve clot specificity and single-chain half-life and reduce the potential for plasminogen activation in the plasma.     

Treatment of acute nonlymphocytic leukemia: use of anthracycline- cytosine arabinoside induction therapy and comparison of two maintenance regimens

Patients with acute nonlymphocytic leukemia were given remission induction therapy consisting of cytosine arabinoside and an anthracycline. Those patients who experienced complete remission received two courses of consolidation therapy and were randomized to receive maintenance therapy consisting of either daily chemotherapy with reinforcements every 3 mo or reinforcement therapy only every 6 wk. The overall complete remission rate was 66%, with 80% complete remission for previously untreated patients less than 60 yr of age who did not have a prior history of malignancy. Remission durations were the same for patients treated with both maintenance regimens. The major determinant for successful remission induction therapy was patient age, with older patients frequently succumbing to intercurrent infection. Documented leukemic cell resistance to the therapy employed was only rarely encountered. Once remission was achieved, age was no longer a determinant of patient survival, since duration of remission was independent of age. Remission durations were directly related to leukemic cell retention of cytosine arabinoside triphosphate. Hence therapy for acute nonlymphocytic leukemia can be divided into two separate areas: remission induction and remission maintenance.     

Repolarization of the membrane potential of blood platelets after complement damage: evidence for a Ca++ -dependent exocytotic elimination of C5b-9 pores

Gel-filtered blood platelets exposed to complement proteins C5b-9 have previously been shown to undergo a reversible depolarization of membrane potential (Em) in the absence of lytic plasma membrane rupture. In this paper, we examine the mechanism by which C5b-9 damaged platelets restore their basal electrochemical state, despite increased ion conductance due to membrane insertion of these cytolytic serum proteins. Repolarization of Em after formation of the C5b-9 membrane pore is shown to be accompanied by a Ca++-dependent vesiculation of the platelet surface, which results in the release of these proteins from the plasma membrane and a restoration of the membrane's functional integrity. This exocytotic elimination of C5b-9 complexes from the plasma membrane is accompanied by a ouabain-inhibitable repolarization of Em, which presumably reflects restoration of transmembrane cation gradients by the plasma membrane Na/K ATPase. The role of external Ca++ in the platelet's response to membrane-insertion of the C5b-9 proteins is discussed both in the context of the known cellular effects of this ion and in the context of recent observations suggesting sublytic changes in platelet function after complement-mediated plasma membrane damage.     

A prospective study of the psychological impact on patients of first diagnosis of human papillomavirus

This study assessed the psychological impact of the first time diagnosis of human papillomavirus (HPV) in consecutive clients attending the Hamilton Sexual Health Clinic, and sought to determine whether this changed over time. Clients with a diagnosis other than HPV and those found to have no diagnosis were compared with HPV clients. All participants completed a battery of questionnaires following their initial visit and again at 4 weeks. The battery consisted of the General Health Questionnaire, Illness Attitude Scales, the International Index of Erectile Function or the Brief Index of Sexual Function for Women, and a 6-question test of the client's knowledge of HPV. One hundred and one participants completed the first questionnaires and 47 of those completed follow-up questionnaires. We found those diagnosed with first episode of HPV had considerable psychological difficulties. However these were no different to those associated with other sexually transmitted infections (STIs) or even those with no active diagnosis.