Hemolysis of human erythrocytes is a new bioactivity of gangliosides.

Using sheep erythrocytes and liposomes, an inhibitory effect of gangliosides has been shown on the activation of the alternative pathway of complement. However, in studies using human erythrocytes, we found that gangliosides had hemolytic activity that was possibly mediated through activation of the alternative pathway. Pretreatment of human erythrocytes obtained from healthy volunteers or paroxysmal nocturnal hemoglobinuria (PNH) patients with a ganglioside mixture purified from human erythrocytes enhanced their susceptibility to homologous human complement, and resulted in dose-dependent hemolysis. The enhancement was more marked in PNH erythrocytes than control cells. Protease treatment of the ganglioside mixture did not change its hemolytic activity, but sialidase treatment abolished the activity. Among the major erythrocyte gangliosides, II3NeuAc-LacCer (GM3) was the most potent hemolytic agent. Gangliosides purified from bovine brain were also active, while neither nonsialylated glycosphingolipids, the ceramide moiety, or sialic acid alone were active. Sialic acid residues in the ganglioside molecules were essential to this activity, but the amount of the residue or the source of the gangliosides seemed not to be important. Several treatments inhibiting the alternative but not classical complement pathway markedly reduced the ganglioside hemolytic activity. This novel bioactivity of gangliosides was thus suggested to be mediated partly by activation of the alternative pathway.

Expression of cryptantigen Th on paroxysmal nocturnal hemoglobinuria erythrocytes in association with a hemolytic exacerbation.

Paroxysmal nocturnal hemoglobinuria (PNH) erythrocytes lack complement regulatory membrane proteins and are susceptible to complement. Although the critical role of complement in intravascular hemolysis in PNH is accepted, the precise mechanism of complement activation in vivo is unknown. Accordingly, in a PNH patient who was suffering from a hemolytic precipitation soon after a common cold-like upper respiratory infection, we analyzed the erythrocytes with lectins and by flow cytometry to detect membrane alteration that lead to complement activation. The lectin reactivity of erythrocytes showed the expression of cryptantigen Th. The patient serum at the time of the hemolysis induced the expression of Th on erythrocytes from PNH patients and from healthy volunteers in vitro, whereas neither the patient serum after recovery from the hemolysis nor blood type-matched control serum from healthy donor showed this activity. Moreover, autologous serum selectively hemolyzed Th+ PNH erythrocytes, but not Th- PNH erythrocytes, or Th+ control erythrocytes. Hemolysis was not observed either in complement-inactivated serum or in blood type-matched cord blood serum, which lacks natural antibodies to cryptantigens. These findings indicate that the immunoreaction of infection-induced Th with natural antibody on PNH erythrocytes is a trigger of the complement activation, leading to intravascular hemolysis.

Altered expression of gangliosides in erythrocytes of paroxysmal nocturnal hemoglobinuria.

In paroxysmal nocturnal hemoglobinuria (PNH), impaired glycosyl-phosphatidylinositol (PI)-anchoring of membrane proteins such as decay-accelerating factor has been known to lead to increased susceptibility to complement. Moreover, abnormal expression of non-PI-anchoring glycoproteins such as C3b/C4b receptor (CR1) or glycophorin-alpha also has been shown in PNH. Therefore, we biochemically analyzed glycosphingolipids (GSL) as one of the membrane glycoconjugates of PNH erythrocytes. Erythrocytes of all seven PNH patients showed altered expression of sialosyl GSL (gangliosides) as compared with the control erythrocytes of healthy donors. Both a sialosylparagloboside (IV6NeuAc-nLc4Cer) among four major gangliosides and some minor gangliosides in normal erythrocytes variably disappeared in erythrocytes from the peripheral blood of PNH patients. As one of the possible mechanisms of altered expression of gangliosides in PNH erythrocytes, structural analysis suggested impaired sialylation of GSL. These results suggest not only the altered metabolism of gangliosides in PNH erythrocytes, but also a metabolic disorder of membrane glycoconjugates as a new feature of PNH.

A new hemoglobin variant. HB Yatsushiro alpha 2 A beta 2 60 Val replaced by Leu.

This study was performed to establish the structural abnormality of a new hemoglobin variant discover-d in a Japanese patient with angina pectoris. The hybridization of the separated hemoglobin with canine hemoglobin revealed a beta-chain anomaly. Peptide betaTp-6 was found to be abnormally located on the peptide map of tryptic digests of the S-carboxymethylated beta-chain from the variant hemoglobin. A structural study on the abnormal betaTp-6 revealed that the variant hemoglobin differs from hemoglobin A by substitution of leucine for valine at residue 60 of the beta-chain. This new variant hemoglobin is designated as hemoglobin Yatsushiro after the name of the city where the propositus lived. The patient is hematologically healthy and his clinical history has nothing to do with this abnormal hemoglobin.

Markedly high plasma erythropoietin and granulocyte-colony stimulating factor levels in patients with paroxysmal nocturnal hemoglobinuria.

In patients with paroxysmal nocturnal hemoglobinuria (PNH), we measured plasma concentrations of endogenous hematopoiesis-regulatory cytokines to characterize bone marrow (BM) hypoplasia which is a major cause of death. Contrary to 10 healthy individuals, all 14 patients with PNH showed increases of erythropoietin (Epo) and granulocyte-colony stimulating factor (G-CSF). There were no signs of infection, renal dysfunction or hypoxia. The lower the hemoglobin level and granulocyte count, the higher the plasma Epo and G-CSF levels. In contrast, marked differences were not found in the levels of interleukin-3 (IL-3), tumor necrosis factor-alpha (TNF-alpha), stem cell factor (SCF), granulocyte/macrophage-colony stimulating factor (GM-CSF), or interferon-gamma) (IF-gamma). The cytokine profiles of PNH patients were quite similar to those of patients with aplastic anemia (AA) and myelodysplastic syndrome (MDS). The cytokine profiles may support a pathological relationship between PNH and these stem cell disorders.

Altered expression of protein kinase C in adult T-cell leukemia cells.

Protein kinase C (PKC) has been shown to be involved in the mitogenic response and in oncogenic cell transformation in many experimental models. We analyzed the expression of PKC in both highly purified leukemic T cells freshly isolated from adult T-cell leukemia (ATL) patients and control T lymphocytes obtained from healthy volunteers. PKC activity was decreased in the ATL cells as compared with the control T cells. Cytosolic PKC activity in the ATL cells was remarkably decreased, whereas particulate membrane PKC activity was similar to the control level. The percentage of PKC activity in the particulate fraction was 34% in the ATL cells and 19% in the control cells. Regarding the altered subcellular localization of PKC activity, phorbol ester-induced translocation of cytosolic PKC was inhibited in some ATL cases. Similarly to the decrease in PKC activity, there was a decrease in the expression of the major PKC isozymes II(beta) and III(alpha) in ATL cells. These results suggest impaired regulation of PKC expression in ATL as well as in many experimental cancers.

Increased plasma decay-accelerating factor levels in paroxysmal nocturnal hemoglobinuria.

Decay-accelerating factor (DAF) is a complement regulatory membrane protein that is often absent from the cell surface of blood cells in paroxysmal nocturnal hemoglobinuria (PNH). DAF has also recently been found in the body fluids of healthy individuals. However, its precise structure and biological significance are not yet clear. To clarify the clinical and pathological implications of free DAF, we measured plasma DAF levels in PNH patients, using a newly developed quantitative enzyme-linked immunosorbent assay (ELISA), with a measurable range between 0.2-12 ng, for soluble DAF. ELISA assays revealed significantly increased plasma DAF levels in PNH patients (258 +/- 150 ng/ml, mean +/- S.D., n = 9) as compared with healthy controls (80 +/- 41 ng/ml, n = 17) (p less than 0.01). Taken together with the finding that DAF is synthesized in and released extracellularly from affected PNH cells, plasma DAF levels would be useful for clinical diagnosis and for the quantitative evaluation of the clonal expansion of affected cells in PNH.

[Serum deoxythymidine kinase activity in adult T-cell leukemia].

Serum deoxythymidine kinase activities (s-TK) of the patients with adult T-cell leukemia (ATL), carriers and healthy persons were measured, using a recently developed TK assay with 125I-iodo-deoxyuridine as a substrate. The mean s-TK values were 3.3 +/- 2.7 U/l (n = 21) in normal subjects (HTLV-1(-)), 4.7 +/- 5.0 U/l (n = 35) in carriers, 9.8 U/l (n = 3) in smoldering ATL and 26.7 U/l (n = 6) in chronic ATL. In the patients with acute ATL, the mean s-TK values before and after chemotherapy were 80.9 U/l (n = 2) and 11.6 U/l (n = 4), respectively. In the follow-up studies of the patients with acute ATL, the changes of s-TK levels revealed earlier than those of serum LDH levels. It is suggested that s-TK activity is more useful than LDH as a parameter of monitoring treatment in ATL.

Hormone-stimulated phosphorylation of liver phosphofructokinase in vivo.

The effect of glucagon on the phosphorylation and the enzymic activity of phosphofructokinase in rat liver in vivo was investigated. Glucagon stimulated the phosphorylation of liver phosphofructokinase approximately 3- to 5-fold and increased cAMP levels 5-fold and blood glucose levels 2-fold over the values obtained for control animals. The specific radioactivity of ATP isolated from liver was the same in both control and hormone-treated animals. During the purification of the 32P-labeled enzyme from both animals, no difference was observed in the total or specific enzyme activities of the enzymes from the various fractions. Thus, phosphofructokinase appears to be phosphorylated in vivo by a cyclic AMP-dependent protein kinase. Although phosphorylation does not affect the maximum catalytic activity of the enzyme, it does render the enzyme significantly more sensitive to ATP inhibition. Thus, at a given concentration of ATP, the phosphorylated phosphofructokinase exhibits considerably lower activity than the unphosphorylated enzyme. The possible relationship between our observations and glucagon-mediated control of glycolysis is discussed.

Cleavage of immunoglobulin G (IgG) and IgA around the hinge region by proteases from Serratia marcescens.

Seven clinical and two nonclinical isolates of Serratia marcescens were examined for their ability to produce extracellular enzymes that cleave immunoglobulin G (IgG) and IgA molecules. All seven clinical isolates excreted a large amount of a 56-kilodalton (kDa) protease (56K protease) and small amounts of a 60-kDa and a 73-kDa protease (60K and 73K proteases, respectively) in culture medium during growth. All purified proteases cleaved IgG and IgA effectively if the level of protease production exceeded 2 to 5 micrograms/ml. The proteolytic activity in the culture supernatant was inhibited by about 85% by a chelating agent (EDTA), which indicated that the major immunoglobulin-cleaving enzyme is the metalloprotease(s) reported previously. Immunological quantification of proteases by single radial immunodiffusion showed similar results: the amount of 56K protease was about 65% and those of the 60K and 73K proteases were about 20 and 5%, respectively. Incubation for 3 h at 37 degrees C was required to generate immunoreactive Fab and Fc fragments. Further analysis of the cleavage products of IgG or IgA demonstrated that the 56K protease, as well as the 60K and 73K proteases, cleaves only the heavy chain of these immunoglobulins near the hinge region to generate Fab and Fc fragments. The susceptibilities of the subclasses of IgG and IgA to the 56K protease were as follows: IgG3 greater than IgG1 greater than IgG2 greater than IgG4 and IgA1 greater than IgA2. IgG2, IgG4, and IgA2 were relatively resistant to the 56K protease.

Adenosine uptake in pyrimidine 5'-nucleotidase deficient human erythrocytes via a high affinity transport system.

Using a pulse-labeling technique, 14C-adenosine uptake into pyrimidine 5'-nucleotidase (P5N) deficient erythrocytes (RBC) was found to be impaired. The Lineweaver-Burk plot showed Km values of 2.0 X 10(-3) mM and 0.2 X 10(-3) mM for normal RBC and P5N deficient RBC, respectively. These results indicate that P5N is one of regulators of the adenosine transport system and/or is associated with adenosine carrier protein.

An interesting syndrome of hemolytic anemia, degeneration of the liver and diabetes associated with a high red cell Mg-ATPase, detected by 31P NMR spectroscopy.

31P NMR was used to study the erythrocytes of three patients who exhibited a familial multisystem disease characterized by fatty liver, diabetes and nonspherocytic hemolytic anemia of unknown etiology. 31P NMR measurements disclosed an abnormally high level of intracellular inorganic phosphate (Pi) and an abnormally low level of ATP in the erythrocytes 6 h after blood withdrawal from proband (I-1). This finding suggested that ATP was markedly decreased in the red cells of this proband, as compared with those of normal subjects. Time-dependent changes of 31P NMR spectra of the erythrocytes from the two daughters (II-1, II-2) of the proband demonstrated clearly an enhanced decomposition of ATP with a concomitant increment of Pi. Several ATP-consuming enzymes in erythrocytes, such as those in the Embden-Meyerhof system, pentose phosphate pathway enzymes, Na+, K(+)-ATPase and Ca2+, Mg2(+)-ATPase, were within normal limits of activity, but Mg2(+)-ATPase was drastically above the normal limit. The Mg2(+)-ATPase activity was 3 times higher in the red cell membranes of these patients than in those from normal subjects.

Inhibition of human immunodeficiency virus infection of human lymphocytes by gangliosides.

As the first step in human immunodeficiency virus (HIV) infection, HIV binds to CD4 molecules on the surface of human T lymphocytes. Expression of CD4 by the cells is remarkably modulated by exogenously added gangliosides, which are normal components of the surface of animal cells. We report here that these physiological molecules also clearly inhibited HIV infection of lymphocytes in vitro in a dose-dependent manner through the selective modulation of CD4 from the cell surface. This raises the possibility of in vivo application of gangliosides as a new strategy for treating acquired immunodeficiency syndrome.

Natural killer activity and antibody-dependent cell-mediated cytotoxicity in multiple myeloma.

Natural killer (NK) activity and antibody-dependent cell-mediated cytotoxicity (ADCC) were studied by routine methods in 11 patients with untreated malignant monoclonal gammopathy. NK and/or ADCC activity was clearly reduced in three patients with advanced disease. Moreover, sera from some myeloma patients impaired the ADCC and NK activity. A large quantity of purified monoclonal IgG from one patient appeared to inhibit NK and ADCC activity as did high concentrations of pooled polyclonal immunoglobulin from healthy persons. In two of these 11 patients, other malignancies were diagnosed prior to chemotherapy. One of these patients, who had nonsecretory myeloma, had marked impairment of NK and ADCC activity; the other, with IgG myeloma, had normal NK and ADCC activity.