Erythrocytes Of Various Species Research Articles

ViroSpot microneutralization assay for antigenic characterization of human influenza viruses

The hemagglutination inhibition (HI) assay has been used for the antigenic characterization of influenza viruses for decades. However, the majority of recent seasonal influenza A viruses of the H3N2 subtype has lost the capacity to agglutinate erythrocytes of various species. The hemagglutination (HA) activity of other A(H3N2) strains is generally sensitive to the action of the neuraminidase inhibitor oseltamivir, which indicates that the neuraminidase and not the hemagglutinin is responsible for the HA activity. These findings complicate the antigenic characterization and selection of A(H3N2) vaccine strains, calling for alternative antigenic characterization assays. Here we describe the development and use of the ViroSpot microneutralization (MN) assay as a reliable and robust alternative for the HI assay. Serum neutralization of influenza A(H3N2) reference virus strains and epidemic isolates was determined by automated readout of immunostained cell monolayers, in a format designed to minimize the influence of infectious virus doses on serum neutralization titers. Neutralization of infection was largely independent from rates of viral replication and cell-to-cell transmission, facilitating the comparison of different virus isolates. Other advantages of the ViroSpot MN assay include its relative insensitivity to variation in test dose of infectious virus, automated capture and analyses of residual infection patterns, and compatibility with standardized large scale analyses. Using this assay, a number of epidemic influenza A(H3N2) strains that failed to agglutinate erythrocytes, were readily characterized antigenically.

Intravascular hemolysis induced by the venom of the Eastern coral snake, Micrurus fulvius, in a mouse model: Identification of directly hemolytic phospholipases A2

Intravascular hemolysis has been described in envenomings by the Eastern coral snake, Micrurus fulvius, in dogs. An experimental model of intravascular hemolysis was developed in mice after intravenous (i.v.) injection of M. fulvius venom. Within one hr, there was prominent hemolysis, associated with a drastic drop in hematocrit, morphological alterations of erythrocytes, hemoglobinemia, and hemoglobinuria. Hemoglobin was identified in urine by mass spectrometry. Histological sections of kidney revealed abundant hyaline casts, probably corresponding to hemoglobin. This effect was abrogated by p-bromophenacyl bromide, indicating that it is caused by phospholipases A2 (PLA2). A monospecific anti-Micrurus nigrocinctus antivenom neutralized hemolytic activity in vivo. When tested in vitro with erythrocytes of various species, a clear difference in susceptibility was observed. Mouse and dog erythrocytes showed the highest susceptibility, whereas human and rabbit erythrocytes were not affected at the experimental conditions tested. The higher susceptibility of dog and mouse erythrocytes correlates with a high ratio of phosphatidylcholine/sphingomyelin in erythrocyte plasma membrane. When mouse erythrocytes were subjected to mechanical stress, after incubation with venom, hemolysis increased significantly, suggesting that both phospholipid hydrolysis by PLA2s and mechanical stress associated with rheological factors are likely to contribute to cell lysis in vivo. Several PLA2s isolated from this venom reproduced the hemolytic effect, and the complete amino acid sequence of one of them (fraction 17), which also induces myotoxicity, is reported. Since very few PLA2s inducing intravascular hemolysis have been described from snake venoms, this enzyme is a valuable tool to identify the structural determinants of hemolytic activity. The mouse model described in this study may be useful to explore the pathophysiology of intravascular hemolysis.

Detection of Nonhemagglutinating Influenza A(H3) Viruses by Enzyme-Linked Immunosorbent Assay in Quantitative Influenza Virus Culture

To assess the efficacy of novel antiviral drugs against influenza virus in clinical trials, it is necessary to quantify infectious virus titers in respiratory tract samples from patients. Typically, this is achieved by inoculating virus-susceptible cells with serial dilutions of clinical specimens and detecting the production of progeny virus by hemagglutination, since influenza viruses generally have the capacity to bind and agglutinate erythrocytes of various species through their hemagglutinin (HA). This readout method is no longer adequate, since an increasing number of currently circulating influenza A virus H3 subtype (A[H3]) viruses display a reduced capacity to agglutinate erythrocytes. Here, we report the magnitude of this problem by analyzing the frequency of HA-deficient A(H3) viruses detected in The Netherlands from 1999 to 2012. Furthermore, we report the development and validation of an alternative method for monitoring the production of progeny influenza virus in quantitative virus cultures, which is independent of the capacity to agglutinate erythrocytes. This method is based on the detection of viral nucleoprotein (NP) in virus culture plates by enzyme-linked immunosorbent assay (ELISA), and it produced results similar to those of the hemagglutination assay using strains with good HA activity, including A/Brisbane/059/07 (H1N1), A/Victoria/210/09 (H3N2), other seasonal A(H1N1), A(H1N1)pdm09, and the majority of A(H3) virus strains isolated in 2009. In contrast, many A(H3) viruses that have circulated since 2010 failed to display HA activity, and infectious virus titers were determined only by detecting NP. The virus culture ELISA described here will enable efficacy testing of new antiviral compounds in clinical trials during seasons in which nonhemagglutinating influenza A viruses circulate.

Comparative analysis of phospholipid composition in blood erythrocytes of various species of mouse-like rodents

Comparative analysis of quantitative phospholipid composition of blood erythrocytes was performed on various species of mouse-like rodents. In erythrocyte phospholipids of common voles there was revealed a relatively low sphingomyelin content, not characteristic of mammals. A hypothesis is put forwards that the unique lipid composition in common vole erythrocytes might be one of adaptation mechanisms for survival of the animals under conditions of pathogenic stress.

Total and free Mg2+ contents in erythrocytes: a simple but still undisclosed cell model.

The concentration of intracellular free Mg2+ ([Mg2+]i) in erythrocytes, measured by means of 31P NMR and using a dissociation constant for MgATP of 38-50 microM, amounted to 0.2 mM [Mg2+]i in the erythrocytes of various species, was not significantly different and was independent of their total Mg2+ content. The more probable value of [Mg2+]i using the more realistic KD of Mg ATP or the null-point method may amount to 0.4 mM [Mg2+]i in erythrocytes is lower than the [Mg2+]i in nucleated mammalian cell types. The lower [Mg2+]i may be caused by a different regulation of Mg2+ influx and Mg2+ efflux by intracellular Mg2+ in erythroblasts. Free and reversibly bound Mg2+ represent a Mg2+ buffer. The main Mg2+-binding substances are ATP and 2,3-bisphosphoglycerate (2,3-BPG). Total Mg2+ content in the erythrocytes of various species is correlated to the concentrations of ATP and 2,3-BPG. The changed Mg2+ level in erythrocytes during deoxygenation, maturation, cold storage, in Mg2+ deficiency and in sickle cell anemia was reviewed.

The sensitivities of various erythrocytes in a haemagglutination assay for the detection of psittacine beak and feather disease virus.

The erythrocytes of various species were tested in psittacine beak and feather disease (PBFD) virus haemagglutination (HA) and haemagglutination inhibition assays to determine which are suitable for use in these assays. HA activity was observed for erythrocytes of the salmon-crested cockatoo, the sulphur-crested cockatoo, the umbrella cockatoo, the goffin's cockatoo and the cockatiel, with differences amongst individuals within species, but not for erythrocytes of humans, the pig, the guinea pig, the chicken, the goose, the rose-ringed parakeet or the budgerigar. Anti-PBFD virus rabbit sera inhibited the virus-induced agglutination of erythrocytes, confirming the specificity of HA activity. This suggests that selection of suitable psittacine species as well as suitable individuals within a species is necessary when obtaining erythrocytes for the PBFD virus HA assay.

Experimental model of autoimmune hemolytic anemia induced in mice with levodopa by intraperitoneal injection or oral feeding

In this paper we describe a murine experimental model of autoimmune hemolytic anemia induced with multiple injections or oral feeding of levodopa. Strain A mice were intraperitoneally injected or fed with levodopa, at a dose equivalent to the one used in human therapy, and subsequently they developed cycles of IgM, IgG and IgA anti-mouse red blood cells (MRBC) autoantibody responses. Levodopa injection induced serum IgM and IgG anti-MRBC responses and levodopa feeding enhanced the serum anti-MRBC IgA response. The appearance of autoantibodies in the serum was followed by binding of the autoantibodies to mice erythrocytes and three phases of anemia. Red cell bound IgM and IgG autoantibodies were predominant in levodopa-injected mice whereas red cell bound IgA autoantibodies were predominant in levodopa-fed mice. The specificity of the serum IgA autoantibody was not restricted since it interacted with erythrocytes of various species.

Acetylated galactosamine is a receptor for the influenza C virus glycoprotein.

We have investigated the specificity of influenza C virus receptor destroying enzyme (RDE) by treatment of erythrocytes of various species with influenza C virus followed by examination of the agglutination patterns of the erythrocytes with a panel of 13 lectins and four anti-human blood group sera of known receptor specificity. Human and animal erythrocytes were agglutinated by lectins SBA, DBA, WFA, VAA II, RCA II, and WGA which have a specificity for the N-acetyl group of galactosamine (NAc-D-Gal) or glucosamine (NAc-D-Gal); this effect was abolished after treatment of erythrocytes with influenza C virus. On the other hand, lectins (RCA I, PNA, APA) with a specificity for D-Gal were able to agglutinate erythrocytes both before and after influenza C virus treatment. Thus, influenza C virus RDE is able to cleave an acetyl group at the 'N' position of galactosamine or glucosamine in addition to acetyl groups in the 'O' position of neuraminic acid and acetylated amino sugars such as galactosamine may act as receptors for the haemagglutinin of influenza C viruses in addition to acetylated neuraminic acid.

Ferrihaemoglobin formation by amyl nitrite and sodium nitrite in different species in vivo and in vitro.

The ferrihaemoglobin (HbFe3+) formation by amyl nitrite (AN) or sodium nitrite (NaNO2) was studied in different species including man, in vivo and in vitro. In in vivo studies AN was administered intravenously (i.v.), intramuscularly (i.m.), by inhalation, or orally. NaNO2 was injected i.v.. AN i.v. produced HbFe3+ much more rapidly than NaNO2 in dogs, cats, rabbits, and rats. In dogs, i.m. injection of AN was followed by a very slow linear increase in the HbFe3+ content. Inhalation of AN did not lead to HbFe3+ formation in dogs unless it was rebreathed in a closed (bag) or not completely open (gas mask) system. HbFe3+ was produced by oral AN in dogs, the effect being enhanced by addition of DMSO. Inhalation of AN by human volunteers in a gas mask and from ampoules crushed close to the nose did not induce haemoglobin oxidation to a practically significant extent, but it was associated with headache, tiredness, dizziness, and a fall in blood pressure. In in vitro studies, in contrast to NaNO2, AN produced HbFe3+ instantaneously in erythrocytes of various species and in purified human haemoglobin. AN 1 mol yielded 2 mol Fe3+. Only 20% of the oxygen released during the oxidation of haemoglobin by AN or NaNO2 was recovered. In 0.2 M phosphate buffer, pH 7.4, 0.01 mol O2/mol AN was consumed. CO2 was released in the presence of AN, but not of NaNO2, from blood, plasma, and 0.02 M NaHCO3 solution. The ratio (lactate)/(pyruvate) decreased when HbFe3+ was formed by AN or NaNO2.

Mouse monoclonal antibodies against bromelain-treated mouse erythrocytes: Reactivity with erythrocytes of various species of animals and idiotypes

Spleen and peritoneal cells from unimmunized BALB/c mice were cultured in the presence of LPS for 24 hr and fused to produce hybridomas secreting antibodies against bromelain-treated mouse erythrocytes (BrMRBC). Three clones from spleen cells and eight clones from peritoneal cells were isolated and characterized further. All the monoclonal antibodies had IgMκ isotype. Their reactivities against untreated and bromelain-treated erythrocytes from various species were assessed by hemolysis and indirect radioimmunoassay; all the clones had similar antigen specificities. On the isoelectric focussing patterns of light chains, they were separated into two groups, two and nine clones, and all the light chains in each group showed identical patterns. The two groups shared no common idiotope detectable by anti-idiotype antibodies prepared by immunization of rabbits with the monoclonal antibodies, but all the antibodies in each group shared common idiotopes. In each group, one antibody had a unique idiotope different from any other antibody, but eight antibodies in a group shared another identical idiotope. These findings suggest the restricted heterogeneity of anti-BrMRBC antibodies in the mouse.

Influenza C virus uses 9-O-acetyl-N-acetylneuraminic acid as a high affinity receptor determinant for attachment to cells.

Identification of the receptor-destroying enzyme of influenza C virus as a specific neuraminate O-acetylesterase has suggested that 9-O-acetyl-N-acetylneuraminic acid is an essential component of the cell surface receptor of influenza C virus (Herrler, G., Rott, R., Klenk, H.-D., Muller, H.-P., Shukla, A. K., and Schauer, R. (1985) EMBO (Eur. Mol. Biol. Organ.) J. 4, 1503-1506). In this report, three common sialic acids, N-acetylneuraminic acid (NeuAc), N-glycollylneuraminic acid (NeuGc), and 9-O-acetyl-N-acetylneuraminic acid (9-O-Ac-NeuAc) were compared for their ability to mediate attachment of influenza A, B, and C viruses to cells. Human asialoerythrocytes were resialylated to contain the three sialic acids in defined sequence on glycoprotein carbohydrate groups using purified sialyltransferases and corresponding CMP-sialic acid donor substrates. While influenza C virus failed to agglutinate native cells or resialylated cells containing NeuAc and NeuGc, resialylated cells containing 9-O-Ac-NeuAc in three different sialyloligosaccharide sequences were agglutinated in high titer. In contrast, most representative influenza A and B viruses examined preferentially agglutinated cells containing NeuAc and NeuGc and failed to agglutinate cells containing 9-O-Ac-NeuAc. Cells containing 9-O-Ac-NeuAc were sensitive to the action of influenza C virus neuraminate O-acetylesterase which converts 9-O-Ac-NeuAc to NeuAc. This treatment abolished agglutination by influenza C while making the cells agglutinable by several influenza A and B viruses. Finally, the ability of influenza C virus to agglutinate the erythrocytes of various species correlated with the presence of 9-O-Ac-NeuAc. The results provide direct evidence that influenza C virus utilizes 9-O-acetyl-N-acetylneuraminic acid as the primary receptor determinant for attachment to cell surface receptors.

Further studies on the role of neuraminidase and the mechanism of low pH dependence in influenza virus-induced membrane fusion.

The role of neuraminidase and the mechanism of low pH dependence in influenza virus-induced membrane fusion have been studied further using fowl plague virus (FPV, H7N1). Two specific anti-FPV neuraminidase antisera obtained from chickens immunized with recombinant virus strains inhibited viral neuraminidase activity without influencing its haemagglutinating activity. These sera totally inhibited the FPV-induced fusion of erythrocytes and partially reduced haemolysis. But both fusion and haemolysis activities could be restored by external addition of Vibrio cholerae neuraminidase, indicating participation of neuraminidase in FPV-induced membrane fusion. With regard to low pH-dependent fusion by influenza virus, it was found that erythrocytes of various species showed different pH optima for haemolysis by FPV and that erythrocytes could be sensitized for fusion and haemolysis by FPV at neutral pH if they had been pretreated with a low pH buffer. These results demonstrated that surface properties of erythrocytes rather than that of the virus are critical in the low pH-dependent fusion and haemolysis by influenza viruses.

Hemolytic factors in Schistosoma japonicum eggs.

Extracts of Schistosoma japonicum eggs were found to exhibit hemolytic activity on erythrocytes of various species. The hemolytic reaction took place more rapidly at 37 degrees C than at 4 degrees C and did not require divalent cations. The degree of hemolysis was dependent on the concentration of the egg extracts. The hemolytic factors seemed to be lipid in nature because of being heat-stable and soluble in chloroform solvent. Further analysis by means of thin-layer chromatography showed that hemolytic activity in the extracts was due to free fatty acids. Analysis by gas chromatography revealed that fatty acids in the egg extracts consist of myristic, palmitic, palmitoleic, stearic, oleic, linoleic, linolenic, and arachidonic acids. Among them, based on the experiments with commercially available fatty acids, arachidonic acid was the most intensively hemolytic, followed by linoleic, linolenic, oleic, palmitoleic, and myristic acids in that order, whereas palmitic acid showed weak activity only in the presence of divalent cations. From the estimated calculation, it was assumed that 1 mg of protein of the egg extracts contains as much as 0.22 mg of free fatty acids.

Release of vesicles containing acetylcholinesterase from erythrocyte membranes by treatment with dilauroylglycerophosphocholine.

The shedding of acetylcholinesterase-enriched vesicles from erythrocytes of various species of animals occurred when cells were treated with C12:0PC. The response was observed shortly after a morphological change of erythrocytes without any accompanying detectable K+ leakage or hemolysis. The vesiculation was inhibited by the presence of serum albumin or by the incorporation of cholesterol into C12:0PC liposomes, indicating that the insertion of C12:0PC into the erythrocyte membrane causes the vesiculation. The ratio of C12:0PC to total phospholipid determined in vesicle fractions was almost the same as that observed in non-hemolyzed cell fractions. This finding suggests that the vesicles were not shed from portions of membranes rich in C12:0PC. The vesicles showed similar characteristics to those generated by ATP depletion; their diameter is 150-200 nm and they are enriched with acetylcholinesterase activity. Erythrocytes became denser when they lost acetylcholinesterase activity on treatment with C12:0PC.

Investigation of the haemolytic activity of Proteus mirabilis strains.

Young broth cultures of all P. mirabilis strains tested exhibited haemolytic activity. This activity seemed to be strongly cell-associated as only a very small fraction of this activity was found in the cell-free supernatant. The haemolysin was only produced by actively growing cells. Inhibition studies with trypsin and chloramphenicol suggested that the haemolysin is of protein nature. Lecithin and serum of several species had an inhibitory effect on the haemolysin. Besides erythrocytes of various species also VERO cells were affected by the haemolysin. A correlation was found between the haemolytic activity of a strain and its virulence in an experimental mouse model.

Hemagglutination of human erythrocytes by uropathogenic E.coli

Adhesion of uropathogenic E.coli strains to periurethral (PU) and uroepithelial cells has been shown to be correlated to a specific mannose-resistant hemagglutination of human erythrocytes (MRHAhum). Freshly isolated uropathogenic and control faecal E.coli strains were therefore investigated for agglutinating capacity of erythrocytes of various species. All (n = 14) pyelonephritic strains agglutinated human erythrocytes but not those of ox or guinea pig. Strains causing asymtomatic bacteriuria and faecal strains from healthy individuals showed a low incidence of this hemagglutinating pattern, and systitis strains an intermediate frequency. In all cases the MRHAhum-property was closely correlated to an increased adhesive capacity to PU-cells. EM findings indicated that pili mediate the adhesion. These pili are not type 1 pili. Thus, the MRHAhum is likely to reflect a specific, surface-associated virulence property of these strains, which may be a prerequisite for the development of pyelonephritis.

Measurements of exit rates to distinguish between facilitated and simple diffusion.

A modification of the method of Sen and Widdas (J. Physiol. London 160:392-403, 1962) was used to measure the rate of exit of several nonelectrolytes from erythrocytes of various species. In spite of additional errors introduced by the larger half-saturation values of the carriers (phi) and concentrations, it was possible to distinguish between systems with small values of phi, systems with relatively large values of phi, and systems involving only simple diffusion. Approximate values of phi in millimoles and of maximum transfer rate (K) in isotonic units per minute were obtained using times and initial slopes measured on experimental curves. The following values in the foregoing units were obtained: human glucose, phi = 1.8, 1.0, K = 0.8, 1.1; human glycerol, phi = 178, 94, K = 4.3, 3.3; sheep thiourea, phi = 56, 56, K = 0.9, 0.6; and rabbit glycerol, phi = 328, 64, K = 2.2, 1.0. Simple diffusion was demonstrated for the following systems: ox-ethylene glycol; ox-glycerol; sheep-ethylene glycol; and sheep glycerol.

Lysolecithin Induced Membrane Alterations in Thymocytes

The effects of lysolecithin and of 2 synthetic ether-desoxy lysolecithin analogs, containing alkyl residues of 16 or 12 carbon atoms, on the agglutination kinetics of calf and rabbit thymocytes by concanavalin A (Con A) were investigated. Unlike the natural lysolecithin, these synthetic analogs are resistant to metabolism by membrane associated enzymes. It was found that pretreatment of thymocytes with lysolecithin or with the C16-analog leads to slightly increased agglutination rates. The C12-analog, in contrast, significantly inhibits thymocyte agglutination by Con A. Moreover, a comparison of these results with lysophosphatide effects on the agglutinability of erythrocytes of various species revealed that the inhibitory effect of the short-chain phosphatide is rather specific for thymocytes. The finding that long- and short-chain lysophosphatides, which have previously been shown to react as adjuvants or immunosuppressants, respectively, induce adserve alterations in thymocyte membranes indicates that these substances may affect the immune response by changing the membrane properties of immune competent cells. Concerning the nature of these membrane alterations it was shown that lysolecithin did not affect the number of Con A receptors per cell nor the affinity of lectin binding. It is therefore concluded that the lysophosphatide induced alterations of Con A agglutinability can not be caused by an uncovering or covering of lectin-receptors.

Antibodies reactive with cell surface carbohydrates.

Normal and immune sera from various animal species were fractionated on columns of Sepharose covalently coupled with the glycoprotein fetuin. Elution of the material bound to fetuin yielded low but reproducible amounts of protein, ranging from 0.02 to 0.2% of the protein mass of the input sera. This material has been identified by immunoelectrophoresis in agar and by zone electrophoresis on cellulose acetate as immunoglobulin. The Ig fractions bound and agglutinated erythrocytes of various species, and also bound to cells from various mouse tissues including heart, kidney, thymus, and spleen. In all cases, the binding was inhibited by glycoproteins such as fetuin and thyroglobulin, by a glycopeptide isolated from fetuin, and by some bacterial lipopolysaccharides. When the binding of these Ig fractions to mouse splenocytes was tested in the presence of 17 saccharides, no inhibition of binding was observed except by sialic acid, D-galactose, N-acetyl-D-glucosamine, and D-mannose, all of which showed partial inhibition. Inasmuch as these four saccharides are present on the carbohydrate moiety of fetuin, the results suggest that the isolated material is a carbohydrate-specific Ig (CS-Ig) fraction of serum capable of binding to the carbohydrate portion of cell surface receptors and glycoproteins. When bound to lymphocytes, these CS-Ig molecules induced redistribution (patching and capping) of cell surface receptors. Moreover, the CS-Ig fractions from chicken and rabbit sera were weakly mitogenic for mouse splenic lymphocytes. CS-Ig fractions are useful new reagents for studying glycoproteins and the interactions and activities of cell surface carbohydrates.

Virus-Erythrocyte Interactions

Publisher SummaryThis chapter summarizes the experimental evidence bearing on the nature of virus-erythrocyte reactions characteristic of several taxonomic groups.. Such evidence is culled from (1) the study of conditions necessary for hemagglutination; (2) the examination of specific factors affecting either the cell or the virion to enhance, alter, or abolish the reaction; and (3) the direct physicochemical analysis of cells, viruses, and “receptor analogs.” The hemadsorption phenomenon also provides evidence for virus-erythrocyte interactions, which is based on the attachment of erythrocytes to infected cells in culture having hemagglutinin at their surfaces. This phenomenon reflects the interaction between erythrocytes and viral envelope components. The major virus groups that react with erythrocytes include myxoviruses, paramyxoviruses, pseudomyxoviruses, adenoviruses, arboviruses, reoviruses, enteroviruses, and miscellaneous hemagglutinating viruse (rubella virus, coronaviruses, rhabdoviruses, and oncogenic viruses). The agglutination of erythrocytes by the direct action of viral particles was first described in connection with myxoviruses. This led directly to the discovery of viral neuraminidase—a property unique to myxoviruses and paramyxoviruses. A number of viruses unrelated to myxoviruses have since been shown to agglutinate erythrocytes of various species. The visible result of viral hemagglutination is the “pattern” formed at the bottom of a test tube or well plate by lattices of red cells lightly conjoined by viral hemagglutinin. Hemagglutination serves as a useful direct means of titering intact viral particles or hemagglutinating subunits.