Mechanism of the effect of sera from rats subjected to high external temperature on rosette formation by splenic lymphocytes from non-immune animals]

Serum of rats subjected to the action of moderately high external temperature increased the immunogenicity of heterologous erythrocytes, but became inactive at a temperature of 2 degrees C and after the treatment with a polyvalent proteinase inhibitor--trasilol. Incubation of sheep erythrocytes with the serum of heated rats intensified the reaction of rosette formation by the lymphocytes of the spleen of intact rats. Incubation of the spleen cells with the sera of intact and heated rats failed to influence the rosette formation. After trasilol treatment the serum of heated rats completely lost the property to intensify the reaction of rosette formation. Phenomenon of intensification of rosette formation was revealed in case of treatment of erythrocytes with the sera at a temperature of 37 degrees C and was not reproduced at a temperature of 2 degrees C. Investigations carried out indicated that the increase in the immunogenicity of sheep erythrocytes and the intensification of their binding with the lymphocyte receptors of intact rats under the effect of the serum of heated animals were caused by the action of the same factor.     

Effects of temperature and bovine serum albumin on lysis of erythrocytes induced by dilauroylglycerophosphocholine and didecanoylglycerophosphocholine.

The effects of the incubation temperature and bovine serum albumin on hemolysis induced by short-chain phosphatidylcholine were examined. The rate of hemolysis of human, monkey, rabbit, and rat erythrocytes by dilauroylglycerophosphocholine showed biphasic temperature-dependence: hemolysis was rapid at 5-10 degrees C and above 40 degrees C, but slow at around 25 degrees C. In contrast, the rate of lysis of cow, calf, sheep, pig, cat, and dog erythrocytes did not show biphasic temperature-dependence, but increased progressively with increase in the incubation temperature. Bovine serum albumin increased the hemolysis of human erythrocytes induced by dilauroylglycerophosphocholine or didecanoylglycerophosphocholine: it shortened the lag time of lysis and reduced the amount of phosphatidylcholine required for lysis. A shift-down of the incubation temperature from 40 to below 10 degrees C also shortened the lag time of lysis of human erythrocytes induced by dilauroylglycerophosphocholine and reduced the amount of phosphatidylcholine required for lysis.     

The titration of tetanus antitoxin I. Factors affecting the sensitivity of the indirect haemagglutination test

Various factors affecting the indirect HA test for the titration of tetanus antitoxin have been evaluated with a view to obtaining maximum sensitivity in tests using unfixed sheep erythrocytes and sheep erythrocytes fixed with glutaraldehyde, formaldehyde and pyruvic aldehyde. The optimal concentration of tannic acid has been found to be 1/40 000 for tanning both fixed and unfixed sheep erythrocytes. Tanned sheep erythrocytes sensitized with 50 Lf/ml of tetanus toxoid at pH 7.2 for one hour were the most sensitive. Although the optimal temperature of sensitization was found to be 56 degrees C, unfixed cells tended to clump and lyse at this temperature. Thus a temperature of 37 degrees C was used to sensitize unfixed sheep erythrocytes. Sheep erythrocytes from different animals and the final concentration of sensitized sheep erythrocytes both had great effects on sensitivity. A final concentration of 0.5% of sensitized sheep erythrocytes was found suitable as a compromise between sensitivity and readability. The loss of sensitivity of fixed and sensitized erythrocytes was investigated by storing these cells at 4-8 degrees C for six to nine months.     

Immunogenic and adjuvant properties of rat erythrocytes subjected to exposure to a high external temperature

The erythrocytes of Wistar rats, subjected to heating at 40 degrees C (4 times, each heating lasting 40 min.) were found to be more immunogenic in mice than the erythrocytes of intact rats. The immunization of intact Wistar rats, in a single injection, with syngeneic erythrocytes obtained from the heated animals did not induce immunological response reaction, whereas 5 injections of these erythrocytes caused an increase in the number of rosette-forming cells. The injection of syngeneic erythrocytes obtained from the heated rats to intact animals also stimulated the development of immune response to sheep erythrocytes.     

A modified theta-toxin produced by limited proteolysis and methylation: a probe for the functional study of membrane cholesterol.

A derivative of cytolytic theta-toxin from Clostridium perfringens was prepared by limited proteolytic digestion of the native toxin followed by methylation. Among the chloroform/methanol-extractable, lipid components of sheep and human erythrocytes, the proteinase-nicked and methylated derivative (MC theta) specifically binds to cholesterol. While MC theta retains binding affinity comparable to that of intact toxin, it causes no obvious membrane damage, resulting in no hemolysis at temperatures of 37 degrees C or lower. Using MC theta, we demonstrated the possible existence of high- and low-affinity sites for theta-toxin on sheep erythrocytes at both 37 degrees C and 10 degrees C. The number of high-affinity sites on sheep erythrocytes was estimated to be approximately 3-times larger at 37 degrees C than that at 10 degrees C. In addition, high- and low-affinity sites were demonstrated in human erythrocytes and a lymphoma B cell line, BALL-1 cells. Both binding sites disappear upon simultaneous treatment of cells with sublytic doses of digitonin, suggesting that cholesterol is an essential component of both the high- and low-affinity sites and that the mode of cholesterol existence in plasma membranes is heterogeneous in these cells. Because of its high affinity for membrane cholesterol without causing any obvious membrane changes at physiological temperatures, MC theta may provide a probe for use in the functional study of membrane cholesterol.     

Hemagglutinating activity inCorynebacterium pyogenes

Thirty-eight strains ofCorynebacterium pyogenes isolated from cases of heifer- and dry-cow mastitis and from other infections of sheep, cows, pigs, and man were screened for agglutination of sheep erythrocytes. Bacteria grown either in serum broth or on blood agar in the presence of CO₂ hemagglutinated. Performance of titrations at 4°C avoided the hemolytic effects ofC. pyogenes. Erythrocytes of cat, chicken, cow, dog, guinea pig, horse, man (Group A), pig, and rabbit were also agglutinated. Pretreatment of sheep erythrocytes with trypsin, pepsin, A1 proteinase or pronase had no effect on agglutinability. Pretreatment ofC. pyogenes with pronase, but not with trypsin, A1 proteinase, or pepsin, abolished hemagglutinating capacity. The hemagglutinin was inactivated by exposure to 60°C for 10 min. Agglutination of sheep erythrocytes was inhibited by five glycoproteins. None of 12 mono-, di-, or trisaccharides nor heparin, chondroitin sulfate, or dextrin inhibited hemagglutination. These data suggest that the receptor may possibly be an oligohexosyl group of a glycoconjugate of lipid nature. Although a few cells of three mastitic strains ofC. pyogenes possessed fimbriae-like surface structures, no correlation between fimbriation and hemagglutinating activity was apparent.     

Occurrence of hemolytic activity in the skin secretion of the caecilian siphonops paulensis.

The skin secretion of the caecilian Siphonops paulensis (SpSS) induces a time-and dose-dependent hemolytic response on red blood cells (RBC). When RBC from various animals species were subjected to the action of SpSS, a range of sensitivities was evident, sheep erythrocytes being the most susceptible, human, mouse and rabbit having moderate susceptibility, cow, snake and toad erythrocytes being more resistant, while S. paulensis RBC were entirely resistant. The hemolytic activity of SpSS was inhibited at temperatures higher than 60 degrees C. Both trypsin- and chymotrypsin-treated SpSS were ineffective in inducing RBC lysis. The treatment of SpSS with sheep RBC ghosts reduced its activity. There is no phospholipase activity in the SpSS.     

Influence of several experimental conditions on the quantity of E- and EAC-rosette forming cells in peripheral human blood]

The authors analysed the influence of some conditions of studying the rosette-formation between the human peripheral blood lymphocytes and sheep red blood cells, not loaded (a) and loaded with antibodies and a complement (b). It was shown that rosette formation depended on the temperature at which the experiment was conducted. Thus, after the incubation of a mixture of cells at 37 degrees C, and then at 4 degrees C the number of rosette-forming cells was greater than after the incubation at 4 degrees C alone. Substances used for their fixation also influenced the formation of rosettes: in the treatment of erythrocytes with formalin, tannin and glutaraldehyde, as well as introduction of the latter into the mixture of erythrocytes and leukocytes considerably increased the number of a and b (the sum of T- and B-lymphocytes exceeded 100%). Among a and b in such experimental conditions there was seen a great number of monocytes and of segmented leukocytes. Fixation of smears prepared from a mixture of cells after the incubation in formalin vapour did not lead to such increase in the number of rosette-forming cells. Apparently in case of using glutardehyde, formalin and tannin cells for the treatment of cells additional investigations are necessary with the purpose of identification of a and b as T- and B-lymphocytes.     

Extracorporeal immunostimulating effect of terrilytin and lysozyme]

The role of splenocytes and erythrocytes in showing an extracorporal action by terrilytin and lysozyme was studied. The extracorporal effect of terrilytin was to a greater extent mediated by the spleen cells adhering to the plastic while the extracorporal effect of lysozyme was mainly mediated by the heavy ("old") erythrocytes. The heat treatment at a temperature of 42 degrees C for 15 minutes did not abolish the terrilytin extracorporal effect mediated by the erythrocytes but completely abolish the similar effect induced by lysozyme which bound to the erythrocyte membrane. After exposure of the erythrocytes to terrilytin, the strength of the lysozyme binding increased and there was a respective increase in the immunostimulating activity of the erythrocytes.     

The effect of febrile temperatures on biologic actions of interferons: abrogation of suppression of delayed-type hypersensitivity and antibody production

Interferons (IFN) have a complex immunoregulatory effect on all cells of the immune system. In most cases in which IFN had an enhancing effect, the suggested mechanism was inhibition of the generation or activity of suppressor cells. In the present study, we examined the effect of IFN on suppression of the delayed-type hypersensitivity (DTH) response. Suppression was induced with a low antigen dose of sheep erythrocytes (SRBC), and IFN was found to abrogate both the suppressed state and the transferability of this state. Cyclophosphamide had the same effect. However, the in vitro generation of suppressor cells was not altered by the addition of IFN to the culture medium at a normal temperature (37 degrees C). To reconcile the disparity between the successful anti-suppressive action of IFN in vivo compared with its failure in vitro, we considered the possibility that the pyrogenic action of IFN in vivo might create the optimal thermal environment for its anti-suppressive action. Indeed, when IFN was then tested in vitro at a febrile temperature (39.3 degrees C), it completely blocked the generation of suppressor cells. On the other hand, once suppressor cells were generated at 37 degrees C, IFN had no effect on their ability to suppress a fresh culture either at 37 degrees C or at 39.3 degrees C. IFN also had no effect on the generation of helper cells at either temperature, but help was greatly enhanced by high temperature alone. In vivo, we found our IFN preparation to be pyrogenic and observed that an anti-pyretic drug given before and during antigen stimulation abrogated the anti-suppressive effect of IFN. We suggest, therefore, that the febrile state induced by IFN promotes its action on suppressor cells.     

C5b-9 assembly: average binding of one C9 molecule to C5b-8 without poly-C9 formation generates a stable transmembrane pore

Membrane attack by serum complement normally results in the formation of C5b-9 complexes that are heterogeneous with respect to their C9 content. We here report that an apparently homogeneous population of C5b-9 complexes can be generated through treatment of C5b-7-laden sheep erythrocytes with C8 and C9 for 60 min at 0 degree C. Experiments performed by using radioiodinated C8 and C9 components have indicated that binding of C8 to these target cells is essentially temperature independent. In contrast, when a surplus of C9 molecules is offered to C5b-8 cells, an approximately fourfold to 4.5-fold higher number of C9 molecules become cell bound at 37 degrees C as opposed to 0 degree C. C5b-9 complexes isolated from target membranes treated with C9 at 0 degree C contain no polymerized C9 and do not exhibit the ring structure characteristic of the classical complement lesion. Nevertheless, these complexes generate stable transmembrane channels and cause hemolysis at 37 degrees C. The pores have been sized to 1 to 3 nm effective diameter by osmotic protection experiments. SDS-PAGE of the isolated complexes indicates an average stoichiometry of only one molecule C9 bound per C5b-8 complex. The results show that oligomerization of C9 with formation of ring lesions is not a basic requirement for the generation of stable transmembrane complement pores in sheep erythrocytes. They indirectly support the contention that terminal complement components other than C9 contribute to the intramembrane domains of C5b-9 pores.     

Effect of temperature shifts on extracellular proteinase-specific mRNA pools in Pseudomonas fluorescens B52.

The influence of a shift in temperature from 20 to 32 degrees C on extracellular proteinase synthesis by Pseudomonas fluorescens B52 was examined. When cells actively synthesizing proteinase at 20 degrees C were shifted to 32 degrees C, enzyme synthesis ceased immediately. After 30 min at 32 degrees C, cells recovered at 20 degrees C after a lag of 30 min. Rifampin and chloramphenicol prevented recovery of synthesis at 20 degrees C. Rifampin-insensitive proteinase synthesis (an indirect measure of proteinase-specific mRNA pools) decreased after the exposure of cells to 32 degrees C for 30 min but was recovered during incubation at 20 degrees C. Controls not exposed to a temperature shift experienced no loss of rifampin-independent synthesis. Cells experienced a 50% reduction in mRNA pools after 15 min at 32 degrees C. The data support the working hypothesis that the loss of mRNA pools after treatment at 32 degrees C is responsible for the lag before the recovery of extracellular proteinase synthesis.     

Effect of temperature shifts on extracellular proteinase-specific mRNA pools in Pseudomonas fluorescens B52

The influence of a shift in temperature from 20 to 32 degrees C on extracellular proteinase synthesis by Pseudomonas fluorescens B52 was examined. When cells actively synthesizing proteinase at 20 degrees C were shifted to 32 degrees C, enzyme synthesis ceased immediately. After 30 min at 32 degrees C, cells recovered at 20 degrees C after a lag of 30 min. Rifampin and chloramphenicol prevented recovery of synthesis at 20 degrees C. Rifampin-insensitive proteinase synthesis (an indirect measure of proteinase-specific mRNA pools) decreased after the exposure of cells to 32 degrees C for 30 min but was recovered during incubation at 20 degrees C. Controls not exposed to a temperature shift experienced no loss of rifampin-independent synthesis. Cells experienced a 50% reduction in mRNA pools after 15 min at 32 degrees C. The data support the working hypothesis that the loss of mRNA pools after treatment at 32 degrees C is responsible for the lag before the recovery of extracellular proteinase synthesis.     

Presence and characterization of complement-like activity in the amphioxus Branchiostoma belcheri tsingtauense

The humoral fluid of Branchiostoma belcheri tsingtauense was examined for the presence of complement-like activity. The humoral fluid showed hemolytic activity for rabbit erythrocytes and those from species representing mammals, birds, amphibians and fish, but not sensitized sheep erythrocytes. There was no relationship between phylogeny of the target erythrocytes and degree of hemolysis. The hemolytic activity was optimally assayed at 20 degrees C, at pH 7.5, and in the presence of 10 mM Mg2+. The hemolytic activity was Mg2+-dependent and heat-sensitive, and was abrogated by treatment with rabbit anti-human C3 serum, zymosan, methylamine, hydrazine, and phenylmethylenesulfonyl fluoride. In addition, Western blotting and titration by turbidimetric immunoassay (TIA) revealed that amphioxus humoral fluid contained C3 component, and its concentration is about 1.17 mg/ml, which is comparable to C3 concentration in human or dog sera. These suggest that the hemolytic activities displayed by amphioxus humoral fluid appear to represent the vertebrate complement system probably operating via the alternative pathway.     

Hemagglutinating activity of trypsinized Clostridium perfringens enterotoxin

Abstract The hemagglutinating activity of Clostridium perfringens enterotoxin (CPE) was studied after trypsin treatment. Untreated CPE did not show any hemagglutinating activity to human type A, B, and O, sheep, chicken, horse, guinea-pig, or rabbit erythrocytes. Trypsinized CPE resulted in a more than 100-fold increase in hemagglutinating activity with rabbit erythrocytes only. Other erythrocytes and trypsinized rabbit erythrocytes were not agglutinated at all. The hemagglutinating activity of CPE was also found on treatment with a lysine-specific proteinase. On the other hand, trypsinized CPE did not significantly increase the cytotoxic and enterotoxic activities. The binding reaction between trypsinized and rabbit erythrocytes was not inhibited by any mono-, di-, or polysaccharides, glycoproteins or ganglioside mixtures. These results suggest that the hydrolysis of bonds involving lysine residues is mainly required for hemagglutinating activity, and that the receptor for trypsinized CPE on rabbit erythrocytes is probably the protein moiety.     

Classification of sheep abomasal mucosal mast cell proteinase as a serine endopeptidase (EC 3.4.21)

1. Diisopropylphosphofluoridate (Dip-F) and phenylmethanesulphonylfluoride (Pms-F) are inhibitors of "serine" proteinases, and L-trans-epoxysuccinylleucylamido-(4-guanido)-butane (E-64) is an inhibitor of "thiol" proteinases. The effects of these inhibitors on sheep mast cell proteinase (SMCP) were examined. 2. Enzyme activity was completely inhibited by 5 mM Dip-F following a 4-hr preincubation period at either 4 degrees C or 30 degrees C but was unusually resistant to the action of 1 mM Dip-F. 3. SMCP activity was inhibited by 1 mM Pms-F at both 4 degrees C and 30 degrees C. Inhibition was reversed by dithiothreitol (DTT), but this effect was virtually eliminated following preincubation with Pms-F at 30 degrees C for 12 hr. 4. SMCP activity was unaffected by E-64. 5. These properties are consistent with the classification of SMCP as a "serine" endopeptidase (EC 3.4.21).     

Involvement of a sialic acid-binding lectin with hemagglutination and hydrophobicity of Flavobacterium psychrophilum

Strains of Flavobacterium psychrophilum were studied for their ability to adhere and cause agglutination of erythrocytes and yeast cells. Strains of the serotype Th showed low or no hemagglutinating (HA) properties toward human, avian, bovine, and rainbow trout erythrocytes, whereas strains of serotype Fd and Fp(T) exhibited distinct HA properties. None of the strains was able to cause agglutination of yeast cells. Greater adherence specificity toward rainbow trout blood cells was seen for the HA-positive strains. Growth at 5 degrees C, compared to that at 15 degrees C, induced an increase in the hemagglutination of some strains. HA activities of F. psychrophilum were inhibited only by sialic acid (N-acetyl-neuraminic acid), heat treatment at 65 degrees C, and proteinase K treatment and not by any of seven other carbohydrates, periodate oxidation, or treatment with trypsin. The supernatant from washed bacterial cells also showed some HA properties. All strains were shown to be highly hydrophobic by the hydrophobic interaction chromatography test, although some contradictions to the results of the salt aggregation test (showing some strains as less hydrophobic) were seen. These results indicate that the aggregation of F. psychrophilum and erythrocytes depend on a lectin present on the surface of HA-positive F. psychrophilum strains and absent on HA-negative strains. This lectin reacts specifically with sialic acid. The adhesion differences observed for F. psychrophilum strains do not appear to correlate with the virulence but still provide insights into the interaction of F. psychrophilum and rainbow trout.     

The indiction by complement of a change in KSCN-dissociable red cell membrane lipids.

During complement lysis of antibody-sensitized sheep erythrocytes (EA) there was a larger loss of membrane phospholipids than during lysis elicited by hypotonic buffer. In addition, membranes prepared from complement-lysed EA had a marked reduction in KSCN (2.4 M)-dissociable membrane cholesterol and phospholipids, as compared to membranes from EA lysed hypotonically. Complement lysis caused a mild reduction in the amount of KSCN-dissociable membrane hexose but no change in the amount of dissociable protein. The impairment in dissociation of membrane lipids was related to the action of C8; it did not occur with membranes from EA that were treated with heat-inactivated (56 degrees C for 30 min) human serum, C4-deficient guinea pig serum, C6-deficient rabbit serum, or the first seven human complement components. EA lysed with limited amounts of complement exhibited a partial impairment in KSCN-dissociable lipids. Membranes from erythrocytes lysed with melittin showed a large increase in dissociation by KSCN of lipids, proteins,and hexoses. Membranes from erythocytes lysed with lysolecithin or phospholipase C showed, in addition to a reduction in dissociable lipid, a much larger reduction in dissociable hexose than a membranes from complement-lysed cells. These profiles of reactivity with 2.4 M KSCN inidcate that the membrane pertubations caused caused by complement may be specific. We conclude that complement-lysis is accompanied by a major rearrangement of membrane cholesterol and phospholipid which could be demonstrated in membranes from cells lysed by only one or very few complement lesions. Therefore, it appears that the lesions induce a propragated change in the lipid organization which extends throughout large areas of the membrane. This change might be responsible for the impairment of membrane permeability that follows the action of complement and results in cell destruction.     

Mechanisms governing the level of susceptibility of erythrocyte membranes to secretory phospholipase A2

Although cell membranes normally resist the hydrolytic action of secretory phospholipase A(2) (sPLA(2)), they become susceptible during apoptosis or after cellular trauma. Experimentally, susceptibility to the enzyme can be induced by loading cells with calcium. In human erythrocytes, the ability of the calcium ionophore to cause susceptibility depends on temperature, occurring best above approximately 35 degrees C. Considerable evidence from experiments with artificial bilayers suggests that hydrolysis of membrane lipids requires two steps. First, the enzyme adsorbs to the membrane surface, and second, a phospholipid diffuses from the membrane into the active site of the adsorbed enzyme. Analysis of kinetic experiments suggested that this mechanism can explain the action of sPLA(2) on erythrocyte membranes and that temperature and calcium loading promote the second step. This conclusion was further supported by binding experiments and assessment of membrane lipid packing. The adsorption of fluorescent-labeled sPLA(2) was insensitive to either temperature or ionophore treatment. In contrast, the fluorescence of merocyanine 540, a probe sensitive to lipid packing, was affected by both. Lipid packing decreased modestly as temperature was raised from 20 to 60 degrees C. Calcium loading enhanced packing at temperatures in the low end of this range, but greatly reduced packing at higher temperatures. This result was corroborated by measurements of the rate of extraction of a fluorescent phosphatidylcholine analog from erythrocyte membranes. Furthermore, drugs known to inhibit susceptibility in erythrocytes also prevented the increase in phospholipid extraction rate. These results argue that the two-step model applies to biological as well as artificial membranes and that a limiting step in the hydrolysis of erythrocyte membranes is the ability of phospholipids to migrate into the active site of adsorbed enzyme.     

Nucleoside transport in human and sheep erythrocytes. Evidence that nitrobenzylthioinosine binds specifically to functional nucleoside-transport sites.

Nitrobenzyl[35S]thioinosine binding and nitro[3H]benzylthioinosine binding to nucleoside-permeable and nucleoside-impermeable sheep erythrocyte membranes was investigated, and compared with that found for human erythrocytes. High-affinity nitrobenzylthioinosine-binding sites (apparent KD congruent to 1 nM) were present on human and nucleoside-permeable but not nucleoside-impermeable sheep erythrocyte membranes (8400 and 18 sites/cell for human and sheep nucleoside-permeable sheep erythrocytes was displaced by nitrobenzylthioguanosine and dipyridamole. Uridine, inosine and adenosine inhibited binding. The smaller number of nitrobenzylthioinosine sites on nucleoside-permeable cells compared with human erythrocytes corresponded to a considerably lower Vmax. for uridine influx in these cells (0.53 X 10(-20) mol/cell per s at 25 degrees C compared with 254 X 10(-20) mol/cell per s). It is suggested that high-affinity nitrobenzylthioinosine binding represents a specific interaction with functional nucleoside-transport sites. The uridine-translocation capacity for each transport site at 25 degrees C is 180 molecules/site per s for both nucleoside-permeable sheep cells and human erythrocytes (assuming a 1:1 interaction between nitrobenzylthioinosine and the nucleoside-transport system).