Pig Red Blood Cells Research Articles

Characterization of Streptococcus suis capsular type 2 haemolysin

The production of a haemolysin by Streptococcus suis capsular type 2 was investigated. Human group O erythrocytes were the most susceptible, followed by horse, sheep, cow and pig red blood cells, which exhibited similar susceptibilities; rabbit erythrocytes were the least susceptible. The haemolysin was produced at the end of the exponential growth phase. The toxin described in this paper was purified by affinity chromatography using a thiopropyl-Sepharose 6B column. It is an extracellular protein with a molecular mass of 65 kDa. The haemolysin belongs to the family of toxins known as antigenically related cholesterol-binding cytolytic toxins, since it shares common characteristics with other members of this family, such as sensitivity to oxygen and oxidizing agents, activation by reducing agents, inhibition by low concentrations of cholesterol, formation of transmembrane pores and a 'multihit' mechanism of action. In addition, anti-streptolysin antibodies inhibited the haemolytic activity caused by the S. suis haemolysin. Antibodies against the haemolysin could not be detected in pigs experimentally infected with a haemolytic positive strain of S. suis capsular type 2. To our knowledge, this is the only Lancefield group D Streptococcus producing a haemolysin with these characteristics. The role of this haemolysin in the pathogenesis of S. suis infections remains to be investigated.

A high efficiency method for purification and assay of bee venom phospholipase A2

Phospholipase A2 (PLA2) is the main allergen of hymenopteran venoms. We describe a highly efficient reverse phase high performance liquid chromatographic method (HPLC) for isolating PLA2 from crude bee venom. This method removes all detectable contaminants such as melittin from PLA2 while preserving the hemolytic action of PLA2. In addition we describe a simple functional assay of PLA2 based on its propensity to cause hemolysis of guinea pig red blood cells. These techniques are particularly well suited to the isolation and assessment of PLA2 of venoms which are available in limited quantities.

Autologous cord blood transfusion

Newborn piglets were exsanguinated (60% of blood volume) and retransfused 1 h later. One test group received adult pig red blood cells, the other piglet cord blood cells; controls were infused with plasma. While all controls died, satisfactory results were achieved in piglets transfused with either adult or foetal blood. The feasibility of collecting human cord blood for transfusion was assessed in 100 samples of human cord blood. Blood was collected aseptically and aerobic and anaerobic cultures set up. All samples of cord blood were sterile, and all were Mycoplasma negative. Coagulation parameters were analysed in eight cord plasma samples stored at -20 degrees C for 45 days. No significant abnormalities were found immediately after birth or after storage.

The dephosphorylation reaction of the Ca(2+)-ATPase from plasma membranes.

The breakdown of phosphoenzyme (EP) of the Ca(2+)-ATPase from pig red blood cell membranes was studied at 37 degrees C by means of a rapid chemical quenching technique. When the enzyme was phosphorylated with [gamma-32P]ATP in media without added MgCl2, all the EP formed disappeared along two single exponential curves, a rapid one with k(app) = 90 +/- 10 s-1 and a slow one with k(app) = 0.7 +/- 0.3 s-1. The amount of EP involved in each reaction was close to 50% of the EP present at the beginning. Only EP of rapid breakdown could account for the steady-state hydrolysis of ATP observed under the same experimental conditions. ADP accelerated the slow reaction 45-fold (k(app) = 31 +/- 9 s-1) with K0.5 = 740 +/- 120 microM as if this reaction represented the decay of CaE1P, which donated its phosphate to water slowly in the forward direction and rapidly to ADP in the reverse direction of the cycle. Combination of Mg2+ with K0.5 = 26.3 +/- 5.0 microM at a single class of site in E1 before phosphorylation increased EP of rapid breakdown at the expense of ADP-sensitive EP so that, at nonlimiting concentrations of Mg2+ in the phosphorylation media, all EP decomposed at high rate. Rapid decomposition was observed even with enough CDTA to chelate most of the Mg2+ remaining from phosphorylation, suggesting that the role of Mg2+ during dephosphorylation was to accelerate the transition CaE1P-->CaE2P, preparing EP for hydrolysis. The combination of ATP at a single class of site with Km = 845 +/- 231 microM accelerated the hydrolysis of CaE2P. Calmodulin alone had no effects on dephosphorylation but enhanced acceleration of hydrolysis of CaE2P by ATP making the decay of EP under these conditions the fastest among those measured. Comparison of the rates of dephosphorylation of EP made in the presence of Mg2+ with those of steady-state Ca(2+)-ATPase activity with and without calmodulin showed that the transition CaE1P-->CaE2P and decomposition of CaE2P by hydrolysis are compatible with their role as obligatory intermediate reactions in the cycle of hydrolysis of ATP by the Ca(2+)-ATPase.

Aspects immuno-hématologiques de la xénotransplantation

The main barrier to pig organ xenograft in humans, is the hyperacute rejection, which is provoked by the fixation of human xenoantibodies on the graft endothelial cells, and by complement activation. Because pig red blood cells and endothelial cells carry common xenogeneic epitopes, human anti-pig xenoantibodies can be detected and titered by pig red blood cells direct agglutination. Individuals of AB blood group have lower anti-pig hemagglutinins titers than those of other blood groups. Anti-A and anti-B alloantibodies do not agglutinate pig red blood cells. The anti-B (and not the anti-A) alloantibodies can be adsorbed on pig red blood cells. A linear B type 2 epitope recognized by human natural antibodies has been previously described on pig cells. If the role of xenoantibodies is prominent in the rejection of pig organ xenografts in humans, the role of alloantibodies remains to be precise.

Characterization of humoral human anti‐porcine xenoreactivity

The presence and specificity of natural xenoreactive human anti‐pig antibodies was studied. Titers of cytotoxic antibodies reactive with porcine blood lymphocytes were similar in sera from healthy controls, patients with high titers of HLA alloantibodies, uremic patients, diabetic uremic patients, kidney allografted patients on maintenance immunosuppressive therapy, patients who had been splenectomized and in bone marrow transplanted patients 6 months after transplantation. Similar titers were also recorded in patients with various autoimmune disorders, such as SLE and RA; there was no correlation to autoantibody titers. Furthermore, we found no correlation to natural isohemagglutinins; absorption with adequate human red cells did not reduce activity. However, absorption with pig red blood cells reduced the titer significantly and absorption with pig platelets completely abolished reactivity. Finally, antibodies were also present in cord blood samples indicating an endogenous ligand as an activating substance for antibody production. Most sera contained only IgM cytotoxic antibodies, but some also had a weak non‐IgM cytotoxic titer. Fluorescence studies with pig blood lymphocytes indicated that even sera that did not contain non‐IgM cytotoxic antibodies had IgG and IgA antibodies reactive with pig cells. Direct immunofluorescence experiments were also carried out to demonstrate the presence of target structures for naturally reactive antibodies on fetal pig islet cells. Pig blood lymphocytes were sensitive to ADCC‐mediated killing by IgG natural antibodies, whereas fetal pig islet cells were resistant. However, using adequate positive controls, such as anti‐pig MHC class I antibodies, it was shown that fetal pig islet cells can be sensitive to ADCC‐mediated killing. Finally, lysostrip and fluorostrip experiments were performed to test whether pig MHC antigens contain target determinants, possibly as oligosaccharide side chains, for natural antibodies. We found that pig MHC class I antigens do not contain such determinants.

Cold activation of Na influx through the Na-H exchange pathway in guinea pig red cells

Previous work showed that amiloride partially inhibits the net gain of Na in cold-stored red cells of guinea pig and that the proportion of unidirectional Na influx sensitive to amiloride increases dramatically with cooling. This study shows that at 37 degrees C amiloride-sensitive (AS) Na influx in guinea pig red blood cells is activated by cytoplasmic H+, hypertonic incubation, phorbol ester in the presence of extracellular Ca2+ and is correlated with cation-dependent H+ loss from acidified cells. Cytoplasmic acidification increases AS Na efflux into Na-free medium. These properties are consistent with the presence of a Na-H exchanger with a H+ regulatory site. Elevation of cytoplasmic free Mg2+ above 3 mM greatly increases AS Na influx: this correlates with a Na-dependent loss of Mg2+, indicating the presence of a Na-Mg exchanger. At 20 degrees C activators of Na-H exchange have little or no further stimulatory effect on the already elevated AS Na influx. AS Na influx is much larger than either Na-dependent H+ loss or AS Na efflux at 20 degrees C. The affinity of the AS Na influx for cytoplasmic H+ is greater at 20 degrees C than at 37 degrees C. Depletion of cytoplasmic Mg2+ does not abolish the high AS Na influx at 20 degrees C. Thus, elevation of AS Na influx with cooling appears to be due to increased activity of a Na-H exchanger (operating in a "slippage" mode) caused by greater sensitivity to H+ at a regulatory site.

Characterization of a natural human antibody with anti-galactosyl(alpha 1-2)galactose specificity that is present at high titers in chronic Trypanosoma cruzi infection.

An antibody reactive with the galactosyl(alpha 1-2)galactose [gal(alpha 1-2)gal] epitope was characterized in human sera by enzyme-linked immunosorbent assay, red blood cell (RBC) and laminin absorption, and oligosaccharide inhibition. This antibody was found evenly distributed between the IgG and IgM classes and was present at high titers in the serum of all normal adults studied, but in 75% of children less than three years of age, it was observed at the lower limit of detection, and gradually increased to adult levels by the age of six. Although this antibody bound to gal(alpha 1-3)gal-linked synthetic antigens, it did not bind to the same residues present in rabbit, rat, and guinea pig RBC or in murine laminin or nidogen. These latter results, plus the fact that antigen-antibody binding was strongly blocked by gal(alpha 1-2)gal but not by methyl-alpha-galactopyranoside or melibiose, suggest that this antibody is indeed different from anti-gal(alpha 1-3)gal antibody. Anti-gal(alpha 1-2)gal antibody levels were significantly elevated in 66% of patients with chronic chagasic cardiomyopathy, but were not elevated in patients with different clinical forms of leishmaniasis, Trypanosoma rangeli-infected patients, or in patients with 15 other infectious and inflammatory diseases. Gal(alpha 1-2)gal antibodies did not absorb to intact T. cruzi parasites, but absorbed strongly to trypomastigote and epimastigote sonicates, suggesting some masking of reactive epitopes.(ABSTRACT TRUNCATED AT 250 WORDS)

Preparative purification of pig red blood cell hexokinase type III using a new efficient chromatographic support.

In this paper we report the purification of pig erythrocyte hexokinase type III, at preparative level, using 52 liters of starting material (hemolysate). This was possible using a new efficient anion exchanger support, the Toyopearl DEAE 650 M which allows completely to change the strategy of removing hemoglobin from hemolysates, permitting to handle large amounts of starting material and reducing work would have required months using conventional anion exchanger supports, to only 2-3 days. Furthermore, we have tested the binding of other red blood cell enzymes to the Toyopearl DEAE 650 M, showing a wider potential use of this chromatographic support for their purification at a preparative level.

Subspecies-specific haemagglutination patterns of fimbriated Bacillus thuringiensis spores

Electron microscopy revealed fimbrial appendages (pili) of three types on the surface of Bacillus thuringiensis spores. Fimbriated spores induced mannose-resistant agglutination of rat, guinea pig and/or chicken red blood cells. Each haemagglutination pattern correlated with the subspecies of a given B. thuringiensis strain, rather than with its flagellar serotype. Fimbriation and haemagglutination patterns of B. thuringiensis spores are considered to be of possible taxonomic value.

Subspecies-specific haemagglutination patterns of fimbriated Bacillus thuringiensis spores

Electron microscopy revealed fimbrial appendages (pili) of three types on the surface of Bacillus thuringiensis spores. Fimbriated spores induced mannose-resistant agglutination of rat, guinea pig and/or chicken red blood cells. Each haemagglutination pattern correlated with the subspecies of a given B. thuringiensis strain, rather than with its flagellar serotype. Fimbriation and haemagglutination patterns of B. thuringiensis spores are considered to be of possible taxonomic value.

Organ distribution of the ro (SS‐A) antigen in the guinea pig

Using an anti-Ro (SS-A)-specific "sandwich" enzyme-linked immunosorbent assay (ELISA) and Western immunoblotting, we determined the Ro (SS-A) content (both quantitative and qualitative) in saline-perfused organs of the guinea pig. All tissue extracts contained substantial concentrations of Ro (SS-A) antigen and could be grouped into 3 categories based on quantitative reactivity in the sandwich ELISA. The 60-kd and 52-kd molecular forms of Ro (SS-A) present in tissue extracts were similar to those described in Wi-L2 extracts, and the 54-kd molecular form of Ro (SS-A) in guinea pig erythrocytes was similar to that found in human erythrocytes. The tissue distribution of the isoforms of Ro (SS-A) was shown by Western immunoblotting to vary in different tissues, and the reactivity to the 60-kd Ro (SS-A) was correlated with the activity seen in the ELISA. Both the 60-kd and 52-kd Ro (SS-A) bands in guinea pig liver extracts were very weak on Western immunoblots, in contrast to the high concentration of Ro (SS-A) antigen in the ELISA. Other data suggest the possible existence of a unique form of Ro (SS-A) in the liver. Guinea pig tissues have 4 Y RNA that are equivalent to the 4 human RNA--hY1, hY3, hY4, and hY5--present in HeLa cells, while guinea pig red blood cells have only one Y RNA, which is equivalent in size to human hY4.(ABSTRACT TRUNCATED AT 250 WORDS)

Erythrocytes modulate the baseline frequency of sister-chromatid exchanges and the kinetics of lymphocyte division in culture

The baseline sister-chromatid exchange (SCE) frequencies of human plasma lymphocyte cultures (PLC), but not pig PLC, were nearly twice as high as those of whole-blood cultures (WBC). Addition of human red blood cells (RBCs) to human PLC decreased the SCE frequency in proportion to the RBC-leukocyte co-incubation interval. When the period of RBC-leukocyte co-incubation was equivalent to the total length of the culture period (72 h), the SCE frequency was similar to that observed in WBC. Shorter co-incubation periods yielded SCE frequencies intermediate between those of PLC and WBC. Regardless of the species, cell proliferation was slower in PLC than in WBC. Experiments where RBCs were added to PLC showed that the time sequence of RBC incorporation also affects the cell-cycle progression of human and pig lymphocytes. When either human or pig RBCs were added immediately after PLC stimulation, the cell-cycle kinetics was similar to that of WBC. Shorter co-incubation periods made cell-cycle progression intermediate between PLC and WBC values. Thus, PBCs modulate the baseline frequency of SCEs in human PLC and the cell-cycle progression of both human and pig lymphocytes in a time-dependent manner. Two possible hypotheses for the heightened frequency of SCEs of human lymphocytes in RBC-free cultures were assessed. The loss of RBC-to-lymphocyte cellular contact in PLC did not influence the SCE frequencies of lymphocytes. Finally, the increase of SCEs in human PLC could not be related to differences in the generation time of lymphocytes in culture.

A New Hemolytic Assay for Bovine Serum Complement and its Application during Experimental Bovine Anaplasmosis

A new hemolytic assay for bovine complement is presented. Using this assay we found a significant reduction in bovine serum complement activity during the acute phase of anaplasmosis, and an increase in the sensitivity of the red blood cells (RBC) to bovine complement lysis in vitro. The new hemolytic test is performed with bovine RBC, rabbit anti-bovine RBC serum and bovine serum complement. An isotonic sucrose Tris-buffered saline solution of ionic strength 0.094 and pH 7.2 was found to be adequate for this test. The titres obtained with this new assay, which uses autologous RBC, are comparable with those obtained using the guinea pig RBC assay. The finding of a reduction in bovine serum complement during anaplasmosis may be suggestive of a mechanism responsible for the pathology of this disease.

Incidence of hemagglutination activity among pathogenic and non-pathogenic Bacteroides fragilis strains and role of capsule and pili in HA and adherence

We analyzed the ability of 120 encapsulated strains of B. fragilis to agglutinate guinea pig and human red blood cells. Sixteen strains showed a strong hemagglutination (HA) ability, 21 strains a moderate HA ability, 7 strains a weak HA ability and 74 strains did not agglutinate the tested red blood cells. Six strains tested from each HA group were able to adhere to cheek epithelial cells and to a cultured human intestinal cell line. Hemagglutinating strains were the most adhesive. By electron microscopy, pilus-like structures were found in three of the encapsulated adhesive strains. Treatment of the bacterial cells with pronase E reduced both HA ability and adherence of piliated encapsulated, and of piliated non-encapsulated strains. Glucosidase treatment of cells reduced HA activity and adherence of piliated encapsulated and of non-piliated encapsulated strains. Finally, it was found that hemagglutinating strains are more frequently isolated from clinical specimens (55%) than from feces of healthy donors (20%).

Incidence of hemagglutination activity among pathogenic and non-pathogenic Bacteroides fragilis strains and role of capsule and pili in HA and adherence

We analyzed the ability of 120 encapsulated strains of B. fragilis to agglutinate guinea pig and human red blood cells. Sixteen strains showed a strong hemagglutination (HA) ability, 21 strains a moderate HA ability, 7 strains a weak HA ability and 74 strains did not agglutinate the tested red blood cells. Six strains tested from each HA group were able to adhere to cheek epithelial cells and to a cultured human intestinal cell line. Hemagglutinating strains were the most adhesive. By electron microscopy, pilus-like structures were found in three of the encapsulated adhesive strains. Treatment of the bacterial cells with pronase E reduced both HA ability and adherence of piliated encapsulated, and of piliated non-encapsulated strains. Glucosidase treatment of cells reduced HA activity and adherence of piliated encapsulated and of non-piliated encapsulated strains. Finally, it was found that hemagglutinating strains are more frequently isolated from clinical specimens (55%) than from feces of healthy donors (20%).

Maintenance of cation gradients in cold-stored erythrocytes of guinea pig and ground squirrel: Improvement by amiloride

Red blood cells of ground squirrel, a hibernator, gain Na at one-third the rate of guinea pig red blood cells when stored in saline medium at 5 °C for several days. This result correlates with the known slower loss of K during storage in ground squirrel cells. In ground squirrel cells Na gain is balanced by K loss, so that there is no net gain of solute; in guinea pig cells the total cation content rises progressively. Amiloride, a drug which inhibits Na entry, retards Na uptake in cells of both species. Surprisingly, amiloride also slowed K loss and, in guinea pig red cells, the decline of ATP content. In guinea pig cells amiloride reduced the gain of total cation by half. The results substantiate the difference in cold sensitivity of ion regulation of red blood cells of these two species and demonstrate the possible usefulness of amiloride-type drugs in nonfreezing preservation of red blood cells.

Differential effects of cooling in hibernator and nonhibernator cells: Na permeation

Unidirectional Na influx is less inhibited by cooling in guinea pig red blood cells than in ground squirrel cells. To isolate the source of this difference, component pathways of 24Na entry were estimated by use of selective inhibitors (ouabain, bumetanide, amiloride). Amiloride slightly inhibited influx in ground squirrel cells at every temperature between 37 and 5 degrees C. Amiloride did not consistently inhibit Na influx at 37 degrees C in guinea pig cells but caused a 44% inhibition at 25 degrees C and a 35% inhibition at 5 degrees C. Cytoplasmic acidification caused an increase in amiloride-sensitive influx in guinea pig cells, which was greater at 20 degrees C than at 37 degrees C; cytoplasmic acidification decreased amiloride-sensitive Na influx in ground squirrel cells at 37 degrees C and had no effect at 20 degrees C.

Erythrocyte receptors for cholera and heat-labile enterotoxins of Escherichia coli

Many scrological reactions using red blood cells (RBC) such as radial immune haemolysis (RIH) and indirect haemagglutination (IH) tests have often been used for the detection of cholera toxin (CT) and heat-labile (LT) enterotoxin produced by porcine and human Escherichia coli strains. In these tests, the enterotoxins bind to sheep, bovine and guinea-pig RBC without any ligand. We studied several factors which might interfere with such binding, as well as the nature of the receptors involved. Treatment of erythrocytes with different enzymes revealed that proteolytic enzymes had no effect on the adsorption of enterotoxins to RBC. Conversely, treatment with neuraminidase increased the adsorption. Experiments carried out with delipidized RBC revealed that none of the enterotoxins under study bound to the cells thus treated. Pre-incubation of ganglioside fractions with the enterotoxins blocked RIH and IH reactions and the biological effect of them on Vero cells. Assaying RBC ganglioside fractions by thin-layer chromatography revealed the presence of GM1. Our results suggest that the receptors for GT and LT enterotoxins in sheep, bovine and guinea pig RBC are gangliosides: mainly GM1.

Erythrocyte membrane ATPase and calcium pumping activities in porcine malignant hyperthermia.

To investigate possible abnormalities in erythrocyte membrane enzyme activities in the pharmacogenetic disorder MH, membrane ATPase activities have been examined in erythrocyte ghosts prepared from red blood cells of MHS and normal swine. While no differences were noted in Mg2+-ATPase activities, the (Na+, K+)-ATPase activity of MHS erythrocyte ghosts was less than that of normal ghosts. Ca2+-ATPase activity exhibited low- and high-affinity Ca2+-binding sites in both types of erythrocyte ghost. While the Km for Ca2+ was greater for normal than for MHS erythrocyte ghosts at the high-affinity Ca2+-binding site, the reverse was true at the low-affinity Ca2+-binding site. Irrespective of the type of calcium binding site occupied, the Vmax for normal erythrocyte ghost Ca2+-ATPase activity was greater than that for MHS ghosts. In the presence of calmodulin, there was now no difference between MHS and normal erythrocyte ghosts in either the Km for Ca2+ or the Vmax of the Ca2+-ATPase activity. To determine if the calcium pumping activity of intact MHS and normal pig erythrocytes differed, calcium efflux from the 45Ca-loaded erythrocytes was determined; this activity was significantly greater for MHS than for normal erythrocytes. Thus, the present study confirms that there are abnormalities in the membranes of MHS pig red blood cells. However, we conclude that these abnormalities are unlikely to result in an impaired ability of MHS erythrocytes to regulate their cytosolic Ca2+ concentration.