Protochordate immunity. I. Primary immune response of the tunicate Ciona intestinalis to vertebrate erythrocytes

Serum from the tunicate Ciona intestinalis contains a low titer, natural agglutinin for a variety of vertebrate erythrocytes. Primary injections of 8.5 × 10³ human erythrocytes or 7.0 × 10² duck erythrocytes into the tunic tissues or perivisceral cavity were agglutinated and then cleared by phagocytosis within 24 hr. Higher concentrations of human or duck erythrocytes injected into the tunic tissues were encapsulated. Concomitant with clearance or encapsulation, serum agglutinin levels decreased and then returned to normal within 24 hr. Tunicates are classified taxonomically with the vertebrates in the phylum Chordata. Since phagocytosis and encapsulation reactions are typical of most invertebrates, the results of this study suggest that tunicate internal defense mechanisms are more closely allied to invertebrates than to vertebrates.     

Effect of cell shape, membrane deformability and phospholipid organization on phosphate-calcium-induced fusion of erythrocytes

Fusion of bovine and goat erythrocytes was studied using the phosphate-calcium protocol. Both bovine and goat red cells are resistant to fusion with phosphate and calcium, under conditions that promote fusion of normal human erythrocytes. Fusion resistance is not related to decreased (5%) membrane deformability of erythrocytes of these species, since chicken erythrocytes which are 40% less deformable than human erythrocytes undergo fusion with efficiency similar to human red blood cells. Incorporation of either phosphatidylcholine or phosphatidylserine into bovine erythrocytes mediated by lipid exchange/transfer protein, caused fusion of these erythrocytes. Fluorescence analysis of merocyanine 540 dye labeled erythrocytes, by flow cytometry, showed that the frequency of cells which exhibit dye binding was much less (35%) in dimyristoylphosphatidylcholine (DMPC) incorporated compared to untreated bovine erythrocytes (80%), indicating that incorporation of DMPC caused closed packing of lipids in the external leaflet of the bilayer. These studies show that fusion of bovine erythrocytes, mediated by phosphate and calcium, has a requirement for either specific phospholipids such as phosphatidylcholine, phosphatidylserine, or closed packing of lipids in the external leaflet of the bilayer.     

Paul-Bunnell antigen and a possible mechanism of formation of heterophile antibodies in patients with infectious mononucleosis

Sera of patients with infectious mononucleosis contain heterophile anti-Paul- Bunnell (PB) antibodies to erythrocytes of numerous mammalian species. Evidence is presented that the corresponding antigen of bovine erythrocytes is not, as previously described, a single molecule, but a series of glycoproteins with glycans terminated with N-glycolylneuraminic acid (Neu5Gc). The latter compound should be an important part of the PB epitope because, in agreement with the results of others, we found that desialylation of the PB antigen abolishes almost completely its activity. We examined three different preparations of GM3 ganglioside for their capacity to bind anti-PB and found that only GM3 from horse erythrocytes containing Neu5Gc exhibited a low although ELISA measurable PB activity. The other two GM3 preparations, from bovine milk and dog erythrocytes, containing N-acetylneuraminic acid (Neu5Ac) bound little if any anti-PB antibodies. This finding confirms a previous report that human erythrocyte Neu5Ac containing sialoglycoprotein with similar O-linked glycans as the PB-antigen of bovine erythrocytes exhibits only very low PB activity (Patarca & Fletcher, 1995, Crit Rev Oncogen., 6: 305). In conclusion, we present a hypothesis that anti-PB antibodies in patients with infectious mononucleosis are formed against infection-induced cell membrane glycoconjugates containing highly immunogenic Neu5Gc.     

Anomeric preference of glucose utilization in human erythrocytes loaded with glucokinase

Human erythrocytes were loaded with homogeneous rat liver glucokinase by an encapsulation method based on hypotonic hemolysis and isotonic resealing. As assayed at 10 mM glucose, glucokinase and hexokinase activities in glucokinase-loaded erythrocytes were 218 and 384 nmol/min/gHb, respectively; whereas hexokinase activity in both intact and unloaded red cells, which contain no glucokinase activity, was about 400 nmol/min/gHb. No difference in the rate of lactate production from glucose anomers between intact and unloaded erythrocytes suggested that the encapsulation procedure itself did not affect glucose utilization in red cells. Alpha-anomeric preference in lactate production from glucose was observed in glucokinase-loaded erythrocytes, whereas the beta anomer of glucose was more rapidly utilized than the alpha anomer in intact and unloaded erythrocytes. The results indicate that the step of glucose phosphorylation determines the anomeric preference in glucose utilization by human erythrocytes, since glucokinase and hexokinase are alpha- and beta-preferential, respectively, in glucose phosphorylation.     

Erythrocyte osmotic fragility and cation concentrations during experimentally induced bovine anaplasmosis

1. The osmotic fragility, the concentrations of Na, K and Ca, the osmolality and the total ATPase activity of bovine erythrocytes from uninfected and Anaplasma marginale-infected bovines were studied in an attempt to correlate these parameters with the decrease in the cellular ATP concentration reported during bovine anaplasmosis. 2. The osmotic fragility found in infected bovine erythrocytes, at 0.52% NaCl, was about two times greater than that observed in non-infected bovines. The increase in osmotic fragility was directly related to the increase in intra-erythrocytic parasitemia. 3. The decrease in ATP concentration reported during bovine anaplasmosis could not be directly related to the increased fragility of these cells. The artificial depletion of erythrocytic ATP did not reproduce the same alteration in the osmotic response to NaCl. 4. The plasmatic and cytoplasmatic concentrations of Na, K and CA did not change significantly during bovine anaplasmosis, whereas the interior of the erythrocytes became hyperosmolal. 5. A. marginale-infected bovine erythrocyte membranes showed an increased ATPase activity when compared to control bovines. Parasite-enriched fractions also presented ATPase activity.     

Encapsulation by hypotonic dialysis in human erythrocytes: a diffusion or endocytosis process

Human erythrocytes subjected to controlled hypotonic dialysis are capable of encapsulating and retaining drugs. Under selected conditions encapsulation has been reported to occur by an endocytosis process. The mechanism by which encapsulation occurs under conditions which are conducive for endocytosis to occur was studied. An analysis of the percentage of cells with endocytic vacuoles was made for cells dialyzed to optimal and suboptimal osmotic pressures for encapsulation. No differences were found with approximately 20% of cells from all preparations containing vacuoles. Transmission electron micrographs of cells in different stages of carrier cell preparation reveal endocytic vacuoles both with and without hemoglobin. However, based on the percentage of exogenous substance encapsulated, encapsulation appears to occur primarily by diffusion and secondarily by endocytosis.     

Amphotericin B encapsulated in micelles based on poly(ethylene oxide)-block-poly(L-amino acid) derivatives exerts reduced in vitro hemolysis but maintains potent in vivo antifungal activity

The core-forming blocks of amphiphilic diblock copolymers based on methoxypoly(ethylene oxide)-block-poly(L-aspartate), PEO-b-p(L-Asp), were derivatized to incorporate stearate side chains. The effects of stearate esterification were assessed in terms of micelle stability and amphotericin B (AmB) encapsulation/release. The level of stearate esterification modulates the relative self-aggregation state of encapsulated AmB as evidenced by absorption spectroscopy. When AmB is physically loaded into polymeric micelles, the onset of hemolytic activity toward bovine erythrocytes is delayed relative to that of the free drug. Furthermore, the extent of esterification (0, 46, or 91%) appears to have profound influence on the time-dependent hemolytic profile of AmB toward bovine erythrocytes. Particularly in the case of highly substituted stearate ester micelles, incomplete and gradual build-up of hemolysis was observed over a period of 24 h. On the basis of the corresponding absorption spectra, we speculate that encapsulated AmB may interact strongly with stearate side chains, resulting in sustained release. In a neutropenic murine model of disseminated candidiasis, kidney colony-forming unit determination revealed dose-dependent efficacy for the polymeric micelle/AmB formulation, which was not significantly different from that of Fungizone at doses of 0.2, 0.3, and 0.6 mg/kg (p = 0.7). Thus, AmB administered via a polymeric micelle formulation retained potent in vivo activity.     

Osmotic properties of bovine erythrocytes aged in vivo

The osmotic properties of bovine erythrocytes aged in vivo were studied by the modified microhematocrit method. The osmotic fragility of older red cells decreases due to their larger relative osmotically non-active volume. Relative critical cell volume of bovine erythrocytes does not alter significantly with cell age. The age dependent change in the osmotic fragility of human red blood cells, the reverse of that found for bovine erythrocytes, is due to a different alteration of the critical cell volume during intravascular erythrocyte aging.     

The nutritional importance of dietary blood components for reproduction in the tsetse fly, Glossina morsitans

Females of Glossina morsitans morsitans were fed diets of different composition and their performance in terms of survival, fecundity and offspring size, used as a basis for assessing the nutritional importance of the dietary constituents for reproduction. Diets comprised defibrinated bovine blood from which individual components had been removed, or various fractions of bovine blood mixed in different ratios and combinations.Flies fed serum-free diets (comprising saline-washed bovine erythrocytes) failed to reproduce and their ovaries were atrophied. Attempts were made to correct these symptoms by adding serum components to washed erythrocytes. Results showed that serum albumin and lipoproteins were vital for ovarian growth. The erythrocytes in defibrinated bovine blood could be replaced by preparations of haemoglobin. Dialysis against isotonic sodium chloride, removal of gamma globulins, removal of lymphocytes or heating to 55°C for 2 h did not alter the nutritional quality of defibrinated bovine blood. The qualitative importance of dietary albumin associated lipids and lipoproteins for oöcyte growth suggests a specialisation which may be peculiar to viviparous Diptera. Results are discussed in terms of the development of synthetic diets for tsetse.     

Improved stability of 2,3-bisphosphoglycerate during storage of hexokinase-overloaded erythrocytes

Human red blood cells were overloaded with homogeneous human hexokinase using a procedure of encapsulation based on hypotonic hemolysis and isotonic resealing and reannealing to achieve a final activity that was 15 times higher than that in control cells. Storage for 5 weeks at 4 degrees C of hexokinase-overloaded erythrocytes shows that these cells undergo small K+ leakage and mean cell volume increase compared with control cells. Furthermore, after these 5 weeks of storage the 2,3-bisphosphoglycerate content was normal while the ATP concentration was slightly reduced. These results and other properties suggest that encapsulation of key glycolytic enzymes in erythrocytes can provide a new way to maintain in vitro functionally active red blood cells for at least 5 weeks.     

Glucose-6-Phosphate Dehydrogenase Activity and Glutathione Stability in Fetal and Adult Bovine Erythrocytes

Previously, we found a substantially higher glucoses-phosphate dehydrogenase (G6PD) activity and a slightly higher 6-phosphogluconate dehydrogenase (6PGD) activity in bovine fetal erythrocytes than in bovine adult erythrocytes (Steensgaard 1968). Now, we have investigated whether these differences in dehydrogenase activities were followed by characteristic differences in glutathione (GSH) stability and glutathione concentration. The results are shown in Table 1, which also gives the results of the same investigations on normal and G6PD deficient human erythrocytes.     

Anaplasma marginale, Eperythrozoon wenyoni: lectin reactions with bovine erythrocytes

Normal bovine erythrocytes were agglutinated with four of five lectins specific for different oligosaccharides. The order of reactivity was wheat germ greater than ricin greater than soybean greater than peanut. Concanavalin A did not agglutinate normal bovine erythrocytes. After neuraminidase treatment of normal bovine erythrocytes, each lectin agglutinated the cells with decreased concentrations of lectin, verifying that partial removal of sialic acid exposes more of each lectin's binding sites or alters the binding site such that fewer molecules of lectin are required to initiate agglutination. A change in agglutination of erythrocytes using soybean agglutinin and peanut agglutinin occurred when cells were obtained from cattle infected with Eperythrozoon wenyoni. The results suggested that an alteration in erythrocyte membranes occurred as a result of this infection as manifested by the increased recognition of both the soybean agglutinin and peanut agglutinin receptor carbohydrates. A similar effect was indicated with erythrocytes obtained during an acute Anaplasma marginale infection; however, an ensuing reticulocytosis masked the effect, requiring the use of fluoresceinated lectins to verify that increased binding of each lectin occurred with infected cells when compared to normal cells.     

Measurement of hemolytic complement and the third component of complement in nonhuman primates.

Complement, determined by hemolytic assay, and the third component of complement (C3), determined by radial immunodiffusion assay, were measured in nine nonhuman primate species. The species studied were the titi (Callicebus mollach). The sooty mangabey (Cercocebus atys), the thick-tailed galago or bushbaby (Galago crassicaudatus panganiensis), the crab-eating monkey (Macaca fascicularis), the rhesus monkey (Macaca mulatta), the bonnet monkey (Macaca radiata), the stumptailed macaque (Macaca speciosa), the yellow baboon (Papio cynocephalus), and the black-and-red tamarin (Saguinus nigricollis). Both sheep and bovine erythrocytes were used in the hemolytic complement assays. With the sheep erythrocyte system, sera from four species (yellow baboon, sooty mangabey, bonnet monkey, black-and-red tamarin) had similar titers with both antibody sensitized and non-sensitized erythrocytes. In contrast, the titers obtained using sensitized bovine erythrocytes was always higher than the values obtained using non-sensitized bovine erythrocytes. In all species, the titers for non-sensitized sheep erythrocytes was higher than the titer for non-sensitized bovine erythrocytes. When the species were compared for cross reactivity using the radial immunodiffusion assay for human C3, the rhesus monkey showed the strongest cross reaction; the thick-tailed galago, a prosimian, showed no detectable cross reactivity; and the other species examined showed intermediate degrees of reactivity.     

Targeting of mycotoxins to reticuloendothelial system of mice with carrier erythrocytes

The tricothecene mycotoxin, T-2 toxin, was encapsulated in bovine erythrocytes for in vivo delivery of T-2 toxin to macrophages. Intraperitoneal injection of bovine carrier erythrocytes (5 X 10(8) cells) containing T-2 toxin saturated mouse liver uptake of erythrocytes by 6 h postinjection. At 24 h postinjection, 20% of the injected carrier cells containing toxin were localized in the liver of mice. Saturation of the liver uptake of bovine carrier cells was independent of encapsulated or free T-2 toxin. A dose of T-2 toxin sufficient to inhibit 50% of the macrophage protein synthesis was targeted to the liver via the carrier erythrocytes. A methodology for delivery of highly toxic molecules to liver macrophages is described.     

Stability of membrane pores in hypotonically dialyzed erythrocytes: coencapsulation of different stokes radius probes can occur for at least 21 days in human erythrocytes

Hypotonic dialysis of human erythrocytes results in porous cell stability for several days. Hypotonic cells stored for 1 week are essentially normal with respect to the preparation of carrier erythrocytes. Afterward, cells begin to irreversibly hemolyze resulting in decreased cell recoveries and decreased encapsulation percentages of two probes, sucrose and inulin. The holes generated by controlled hypotonic dialysis (100 mOsm/kg) are unlike the single rupture hole generated by dialysis to 10-20 mOsm/kg. The minimum pore size of resealed, annealed carrier cells is confirmed to be less than 5.2 A.     

The Sedimentation Pattern of D-Glucose-6-Phosphate Dehydrogenase from Bovine Fetal and Adult Erythrocytes

Erythrocytes from bovine fetuses contain about 2.4 times higher D-glucose-6-phosphate dehydrogenase activities than erythrocytes from adult cows and bulls. Studying whether this is due to the existence of a special fetal type of enzyme or an increased amount of enzyme in fetal erythrocytes, the sedimentation coefficients of the enzymes have been estimated by s-zonal ultracentrifugation, and compared to normal and deficient human erythrocyte D-glucose-6-phosphate dehydrogenase, s-zonal ultracentrifugations have been performed with a computer optimized isokinetic sucrose gradient. The mainlines in the program used for calculation of sedimentation coefficients are described. Bovine fetal and adult erythrocyte D-glucose-6-phosphate dehydrogenase was found to have the same sedimentation coefficient of 7.4 S which is different from the sedimentation coefficient of 6.4 S of both human types of the enzyme. The sedimentation coefficients of 6-phospho-D-gluconate dehydrogenase from bovine fetal, bovine adult and human erythrocytes were 6 S for all three types of this enzyme. By cellulose acetate electrophoresis bovine fetal and adult D-glucose-6-phosphate dehydrogenase show the same mobility, again differing from the normal and deficient human type. The results of these experiments show that bovine fetal and adult erythrocytic D-glucose-6-phosphate dehydrogenase with respects to molecular parameters are closely related and perhaps identical enzymes.     

A Rapid Method for Determining T Lymphocyte Levels Using Acid Alpha-Naphthyl Acetate Esterase

A method for rapidly determining levels of T lymphocytes in humans is presented. The method involves staining lymphocytes in peripheral blood or buffy coat smears for acid alpha-naphthylacetate esterase for 1 hr at 37 C. After counterstaining with Wright's stain, the percentage of esterase positive cells is quantitated microscopically. The entire procedure takes less than 2 hours. Data are presented which show that the percentage of esterase-positive lymphocytes accurately reflects the percentage of lymphocytes forming E rosettes with sheep erythrocytes.     

In vitro adherence of bovine erythrocytes infected with Babesia bovis to thrombospondin and laminin

This study was undertaken to examine how eight putative adhesive agents bound to plastic surfaces affected the capacity of bovine erythrocytes, infected with either virulent or avirulent strains of Babesia bovis, to adhere in vitro. Thrombospondin (TSP) induced B. bovis-infected bovine erythrocytes to adhere and adherence was augmented when the infected blood was cultured for 24 h before the assay. Moreover, TSP also caused erythrocytes infected with avirulent strains of B. bovis to adhere to plastic in vitro. Laminin promoted the adherence of infected, and to a lesser extent, of uninfected erythrocytes.     

A study of the candidate virulence factors of Bacteroides fragilis

Bacteroides fragilis strains were classified as virulent or avirulent on the basis of their clearance from the subcutaneous tissues of mice. To determine the factors which may contribute to the virulence of B. fragilis strains, we studied encapsulation, hydrophobicity, growth rate, serum sensitivity, agglutination with erythrocytes of different origin, and neuraminidase production. The strains of the virulent group displayed a higher growth rate in broth and a lower sensitivity to the bactericidal activity of serum than the strains of the avirulent group. They also agglutinated different types of erythrocytes more strongly than did the avirulent strains. No significant differences were found between the two groups of strains as regards encapsulation, hydrophobicity and neuraminidase activity.     

Differential adhesion of major surface proteins 1a and 1b of the ehrlichial cattle pathogen Anaplasma marginale to bovine erythrocytes and tick cells

Anaplasma marginale is a tick-borne ehrlichial pathogen of cattle for which six major surface proteins (MSPs) have been described. The MSP1 complex, a heterodimer composed of MSP1a and MSP1b, was shown to induce a protective immune response in cattle and both proteins have been identified as putative adhesins for bovine erythrocytes. In this study the role of MSP1a and MSP1b as adhesins for bovine erythrocytes and tick cells was defined. msp1alpha and msp1beta1 genes from the Oklahoma isolate of A. marginale were cloned and expressed in Escherichia coli K-12 under the control of endogenous and tac promoters for both low and high level protein expression. Expression of the recombinant polypeptides was confirmed and localised on the surface of transformed E. coli. The adhesion properties of MSP1a and MSP1b were determined by allowing recombinant E. coli expressing these surface polypetides to react with bovine erythrocytes, Dermacentor variabilis gut cells and cultured tick cells derived from embryonic Ixodes scapularis. Adhesion of the recombinant E. coli to the three cell types was determined using recovery adhesion and microtiter haemagglutination assays, and by light and electron microscopy. MSP1a was shown by all methods tested to be an adhesin for bovine erythrocytes and both native and cultured tick cells. In contrast, recombinant E. coli expressing MSP1b adhered only to bovine erythrocytes and not to tick cells. When low expression vectors were used, single E. coli expressing MSP1a was seen adhered to individual tick cells while reaction of tick cells with the E. coli/MSP1a/high expression vector resulted in adhesion of multiple bacteria per cell. With electron microscopy, fusion of E. coli cell membranes expressing MSP1a or MSP1b with erythrocyte membranes was observed, as well as fusion of tick cell membranes with E. coli membranes expressing MSP1a. These studies demonstrated differential adhesion for MSP1a and MSP1b for which MSP1a is an A. marginale adhesin for both bovine erythrocytes and tick cells while MSP1b is an adhesin only for bovine erythrocytes. The role of the MSP1 complex, therefore, appears to vary among vertebrate and invertebrate hosts.