Adhesion of sickle neutrophils and erythrocytes to fibronectin.

The pathophysiology of vaso-occlusive crisis in sickle cell disease involves interactions among blood cells, plasma proteins, and vessel wall components. The initial goal of this work was to quantify the adhesion of sickle red blood cells (RBCs) to fibronectin immobilized on glass under both static and dynamic shear stress conditions. High-power microscopic inspection of static assay plates showed striking numbers of adherent neutrophils as well as RBCs. Sickle neutrophils and RBCs were significantly more adherent to fibronectin than the corresponding normal cells in static adhesion assays. Adhesion of both sickle neutrophils and sickle RBCs in dynamic adhesion assays was promoted by a period of static incubation preceding initiation of shear stress conditions. Adherent neutrophils remained attached at shear stresses up to 51 dyne/cm2; most adherent RBCs were attached at shear stresses up to 13 dyne/cm2, but detached at a shear stress of 20 dyne/cm2. Sickle neutrophil adhesion was enhanced significantly by autologous plasma. Elevated levels of plasma interleukin-6 (IL-6; but not IL-1 or IL-8) were found in 6 of 9 sickle cell disease samples examined, and elevated levels of tumor necrosis factor were found in 2 of 9 samples. Plasma IL-6 levels correlated positively with both the number of sickle neutrophils adherent to fibronectin and the ability of sickle plasma to enhance adhesion of normal neutrophils to fibronectin. These data suggest possible roles for neutrophil activation and for fibronectin in mediating sickle neutrophil and RBC adhesion.

Influence of pH and temperature on hemolysis by adult Schistosoma mansoni membranes.

Membrane fractions from homogenized adult Schistosoma mansoni are known to lyse host red blood cells (RBC's), which serve as an important nutrient source for the parasite. In order to learn more about the hemolytic process, we investigated the effects of pH and temperature on the steps involved in the hemolytic process. For maximum schistosome induced hemolysis to occur the worm lytic agent must be in contact with RBCs in a low pH (pH 5.1), high temperature (37 degrees C) environment for a short time (30 min), after which hemolysis occurs at both pH 7.5 and 5.1. At pH 7.5 the hemolytic process is relatively temperature independent and highly concentration dependent. Dose-response experiments suggest that a multi-hit process of hemolysis is probably involved. Temperature and dextran experiments suggest that a pore is formed in the RBC membrane at pH 7.5. At pH 5.1 hemolysis is temperature dependent and not very concentration dependent. Dose-response data suggest that a single-hit process of hemolysis is utilized at low pH. The hemolytic process at pH 7.5, the pH of the host blood, and pH 5.1, the approximate pH of the worm gut, appears to be very different.

Schistosoma mansoni: membranes from adult worms reversibly perturb shape, volume, and membrane organization of intact human red blood cells.

Adult forms of Schistosoma mansoni ingest host (human) red blood cells (RBCs). To elucidate potential mechanisms by which contact with adult parasites perturbs RBC membranes, we studied the effects of the membrane fraction of isolated schistosomes on RBC shape, volume, potassium ion content, and phospholipid and transmembrane protein lateral mobility. S. mansoni-treated RBCs exhibited rapid but spontaneously reversible shape change from discocytes to spheroechinocytes, reversible decrease in cell volume, and rapid loss of intracellular potassium ions. Treated RBCs also showed rapid but spontaneously reversible immobilization of membrane phospholipids and of band 3, the major transmembrane protein. These data suggest that components of adult S. mansoni membranes can perturb host RBC volume and membrane organization. In the absence of RBC lysis, RBC metabolic and repair mechanisms can reverse these effects.

Lytic activity from schistosomes: interactions with blood cells.

1. The schistosome lytic agent hemolyzed animal red blood cells (RBCs) containing high concentrations of membrane phosphatidyl choline (dog, mouse, and rat) more efficiently than RBCs having no phosphatidyl choline (goat and sheep). 2. Human mononuclear cells lost viability in the presence of the schistosome lytic agent. 3. Preincubating the lytic agent with phosphatidyl choline or bovine serum albumin reduced its lytic activity. 4. Extracellular albumin protected the RBCs from schistosome induced hemolysis. 5. Pretreatment of the RBCs with various proteases enhanced lysis by 10-30%.

Methods for the detection of Jk heterozygotes: interpretations and applications.

The lytic properties of red cells from Jk(a+,b+), Jk(a-, b+) and Jk(a+b-) (normal), Jk(a-b-), and obligate Jk heterozygotes were studied. The Jk(a-b-) cells did not hemolyze for at least 15 minutes in either 2 M urea or methylurea, whereas normal cells were completely hemolyzed in 2 minutes. Red cells from Jk heterozygotes demonstrated intermediate levels of hemolysis when compared to normal and Jk(a-b-) red cells. In addition, these cells had less than 10 percent hemolysis when suspended in 2 M methylurea prepared in 0.4 percent phosphate-buffered saline (pH 7.2). This method may be an alternative to hemagglutination titration scoring for the detection of Jk, especially in paternity cases.

Comparative effects of sugars on the hemolytic activity of Schistosoma mansoni and other hemolytic agents.

1. The rate of red blood cell lysis by the hemolytic agent in adult worm homogenates of Schistosoma mansoni is slowed in the presence of added sugars (50 mM). 2. Trisaccharides were the most effective in slowing and reducing lysis. Disaccharides were more effective than monosaccharides. 3. The addition of sodium, potassium or lithium chloride salts (25 mM) stimulated hemolysis by the S. mansoni agent. 4. Hemolysins with known mechanisms were tested to determine the effects of added sugars (50 mM) or salts (25 mM). 5. The S. mansoni hemolytic agent responds to the addition of sugars and salts in a manner similar to small membrane pore formers.

The effect of 2 molar urea on Jk (a-b-) red cells.

The phenomenon of red cell lysis in urea was investigated for both normal and Jk(a-b-) red cells. Using 14C urea, urea was shown to be transported across the membrane in both cell types. In normal cells this resulted in an influx of water, cell swelling and complete lysis within 2 min. However, in Jk(a-b-) red cells, the presence of urea above 300 mM slowed water movement and delayed hemolysis for at least 15 min. Similar effects could be induced by pretreating normal red cells with p-chloromercuribenzene sulfonate (PCMBS) or by raising the pH of the urea solution to 8.0.

Schistosoma mansoni: activation of hemolytic activity in homogenates from live adult worms.

We have previously described hemolytic activity in extracts of lyophilized Schistosoma mansoni adult worms. In contrast, freshly homogenized live worms have little hemolytic activity. However, preincubation of the homogenate at 38 C, pH 5.1 for 22 hr resulted in an 18 fold increase in specific activity (Hb released/mg protein) due to dramatic increases in hemolytic activity and decreases in protein concentration. No activation occurred when the homogenate was boiled prior to preincubation or when preincubation was performed at 4 C. In addition, a thermostable inhibitor of hemolytic activity is present in freshly homogenized live adult S. mansoni. Hydrolysis of the inhibitor and activation of the hemolytic agent appear to be due in part to hydrolytic activity of one or more cysteinyl proteinases.

Schistosoma mansoni: characterization of hemolytic activity from adult worms.

Homogenates of adult Schistosoma mansoni worms contain a hemolytically active component(s). Centrifugation at 10,000 g shows the major activity is present in the pellet fraction. Red blood cell lysis with the schistosome hemolytic agent is optimal at acid pH (5.0) and highly temperature dependent. The hemolytic component is resistant to boiling (5 min) and stable for extended periods of time at 38 C (22 hr). The length of the lag phase prior to hemolysis and the rate of hemolysis are both concentration and temperature dependent. Following hemolysis, red blood cell ghosts remain.

Adenylate cyclase in adults and cercariae of Schistosoma mansoni.

Adenylate cyclase of adult Schistosoma mansoni is activated by serotonin (5-hydroxytryptamine). Serotonin activation is markedly enhanced by GTP. the poorly hydrolyzed analogs of GTP, guanylyl imidophosphate (Gpp(NH)p) and guanosine 5' -(3-0-thio)triphosphate activate the schistosome cyclase in the absence of serotonin. The activating effect of these nucleotides is increased in the presence of serotonin. Female adult schistosomes have only 40% as much NaF-stimulated activity as do the male parasites. In addition, the serotonin stimulation of adenylate cyclase in the females is only 20% as much as the males. Maximal activation of adenylate cyclase with NaF reveals that the adult parasites have tenfold higher activity than the cercariae. cercarial adenylate cyclase shows very poor response to serotonin in the presence of GTP. Serotonin caused more significant activation of cercarial adenylate cyclase in the presence of guanylyl imidophosphate.

Recovery of Chinese hamster cells from mercuric chloride exposure.

The effects of chronic treatments with HgCl2 on cell survival, DNA replication, and cell progression in cultured Chinese hamster ovary cells were investigated. The ability of these cells to recover from the effects was also characterized. Exposure of cells to 4 X 10(-5) M HgCl2 for 30 min killed about 50% of the cells, and this proportion did not increase with continued exposure up to 24 h. The rate of DNA replication was reduced to 28% of the control rate in the presence of HgCl2. However, when the cells were returned to medium without HgCl2, the rate of DNA replication recovered to 88% of control after 3 h of exposure and 55% of control after 8 h of exposure. The cell doubling time was increased from a control value of 16 h to 31 h in the presence of HgCl2. When the exposed cells were returned to medium without HgCl2, the doubling time returned to 16 h. The rate of progression of cells from G1 phase to S phase was greatly reduced in the presence of HgCl2, and no recovery was observed in this case when the cells were transferred to normal medium. These findings suggested a correlation between ability to recover and the cytotoxic effects of HgCl2.

Effects of mercuric chloride on synchronized Chinese hamster ovary cells: survival and DNA replication.

Chinese hamster ovary (CHO) cells in vitro were treated with HgCl2 at various stages in the cell cycle and the effects of this chemical on cell survival, DNA replication, and cell division were observed. In terms of survival the early G1 cells were the most sensitive to treatment, followed by late G1 and early S, while mid S and late S-G2 treated cells were the least sensitive. Treatment with HgCl2 also resulted in reduced rates of DNA replication and delays in cell division. The early G1 treated cells showed substantially reduced rates of DNA replication followed by 4--5 h division delay. The early S and late S-G2 treated cells had some reduction in their rates of DNA replication followed by corresponding division delay of 2.5 h in the early S treated cells and 1 h in the late S-G2 treated cells.