The Conglutination Phenomenon: XIII. In vivo Interactions of Conglutinin and Experimental Bacterial Infection

Some interrelations between the conglutinating activity of the serum of an animal and experimental bacterial infection were investigated. The passive transfer of conglutinating activity was demonstrated. The level of this activity reaches a peak within 2 days after subcutaneous injection, then declines until no significant titres are demonstrable in 12 to 14 days. It is shown that infection of animals with Salmonella typhimurium causes a rapid reduction in the conglutinating activity of the serum immediately after challenge. Evidence is presented which indicates that the previous injection of conglutinin preparations enhances bactericidal activity of the mouse against Salm. typhimurium.     

The Relationship of the Haemolytic Activity of Complement to the Concentration of Sensitized Erythrocytes

The relationship of the haemolytic activity of complement (C′) to the concentration of sensitized erythrocytes (EA) has been found subject to precise mathematical definition, on the basis of data obtained from simultaneous C′ titrations carried out with twelve different EA concentrations ranging from 10⁷ EA/ml. to 2×10⁹ EA/ml. Increase in EA concentration is accompanied by increase both in the amount of C′ required for 50 per cent haemolysis (RD₅₀ C′), and in the value of σ (reciprocal of the slope of the curve relating probit percentage haemolysis to log C′). The relationship of RD₅₀ C′ to EA concentration is an exponential one; the equation expressing this relationship contains a parameter which is interpreted as consistent with the existence of a threshold value of C′ which should be taken into account in assessing RD₅₀ C′. An explanation of the increase in RD₅₀ C′ and σ with increase in EA concentration is offered.     

The Conglutination Phenomenon: XIV. The Resistance Enhancing Effect of Conglutinin and Immuno-Conglutinin in Experimental Bacterial Infections

The effect of conglutinin and immuno-conglutinin preparations on the resistance of animals has been studied in seven bacterial infections. In six infections —Salmonella typhimurium, Pasteurella septica, Klebsiella pneumoniae, Listeria monocytogenes, Streptococcus pneumoniae and Streptococcus pyogenes—higher survival rates were demonstrated in mice previously injected with the conglutinin preparations.

Infection with avirulent strains or non-pathogenic organisms in numbers large enough to cause death led to severe toxaemia against which the injection of conglutinin preparations failed to protect. Although the conglutinin preparations failed to protect against avirulent or unadapted strains, they did protect against virulent or adapted strains of the organisms.

Experiments undertaken to define the protective factor in the serum preparations indicate that the protective factor probably is the same as that which is responsible for the conglutinating activity, conglutinin.

     

Studies of the Reaction Between the Sensitized Erythrocyte and the First Component of Complement

When sensitized erythrocytes (EA) possessing the 1st (C′₁), 4th (C′₄) and 2nd (C′₂) components of haemolytic complement were treated with 1,4-butanediamine, C′₁ but not C′₄ or C′₂ was removed from the cell. All the straight chain diamines of 3 to 8 carbons, with the amino groups on either end, removed C′₁ with about equal facility; the diamines were considerably more active than the corresponding monoamines. C′₁ could be recovered in solution following its removal from the red cell by the diamine. The rate of detachment of C′₁ was quite high for the first 10 minutes, after which it was slower and followed first order kinetics. It appears that C′₁ and the diamine compete for the same site on the sensitized erythrocyte. The following observations suggest that Ca⁺⁺ has a role in the EAC′₁ bond: (a) An increased Ca⁺⁺ concentration diminishes the effect of the diamine. (b) The EAC′₁ bond dissociates at low bivalent cation concentration. (c) A number of bivalent cations will preserve the EAC′₁ bond, Ca⁺⁺ being the most effective.     

The Specificity of Alternative Complement Pathway-Mediated Lysis of Erythrocytes: A Survey of Complement and Target Cells from 25 Species

Sera from 20 species of mammals were tested for their ability to lyse erythrocytes from 18 species of mammals and birds by the alternative complement pathway. Erythrocytes were not lysed by homologous complement, with one minor exception, but all erythrocytes tested were lysed by at least one complement source, and all sera tested except that of the horse lysed at least one type of erythrocyte. Control experiments indicated that lysis was via the alternative complement pathway and that antibodies were not involved. Complement from the various species could be ranked from most active to least active, and erythrocytes could be ranked from most susceptible to least susceptible. There was an inverse correlation between complement activity and erythrocyte susceptibility. The ranking of the orders of placental mammals, from strongest to weakest complement, was carnivore>oartiodactyl (ruminants and swine) > primate = armadillo > rodent > rabbit > horse. Opossum serum had activity that placed it in the centre of this range. Ferret complement, the most potent tested, lysed all erythrocytes tested except for homologous erythrocytcs, with APCH50 titres as high as 4000. Although the overall reactivity pattern was clear, there were several striking exceptions. For example, the only complement source which lysed ferret erythrocytes was sera of the mouse. The amount of sialic acid present on erythrocytes of 14 mammals was determined, and was, in general, directly correlated with resistance to alternative complement pathway lysis, although there were prominent exceptions to this correlation, involving crythrocytes of the horse, burro and human. All 20 types of complement were also tested for their ability to lyse antibody-coated human tumour cells, under conditions in which both the classical and alternative complement pathways were functional. The data obtained suggest that alternative pathway activation is, in some cases, a major factor determining the effectiveness of a particular complement source in the lysis of xenogeneic tumour cells.     

Visceral Leishmaniasis: I. Unusual Interrelationships Between Immature Erythrocytes and Both Parasites and Mature Erythrocytes

To the Editor Ultrastructural Pathology     

Visceral Leishmaniasis: I. Unusual Interrelationships Between Immature Erythrocytes and Both Parasites and Mature Erythrocytes

To the Editor Ultrastructural Pathology     

Destruction of Sensitized Erythrocytes by Human Monocytes in Vitro: Effects of Cytochalasin B, Hydrocortisone and Colchicine

The purpose of this study was to characterize the destruction of sensitized erythrocytes by human blood monocytes in vitro The incubation in vitro of human monocytes with ⁵¹Cr-lalbelled human erythrocytes sensitized with IgG rhesus alloantibodies anti-D EAIgG anti-D) resulted in release of⁵¹Cr from the erythrocytes (lysis) as well as uptake of ⁵¹Cr-labelled erythrocytes by the monocytes (phagocytosis). The lysis of EAIgG anti-D by monocytes was not dependent on phagocytosis, because cytochalasin B, which inhibited phagocytosis of EAIgG, enhanced lysis. In contrast, hydrocorlisone and colchicine inhibited lysis, but had no effect on phagocytosis. These agents did not affect binding of EAIgG anti-D to monocytes. The effect of these agents on lysis corresponded to their effect on release of lysosomal enzymes by monocytes. The release of lysosomal enzymes, when induced by EAIgG anti-D, was, likewise, enhanced by cytoehalasin B and inhibited by hydrocortisone and colchicine. A significant correlation was found between lysosomal enzyme release and lysis. Together, these results strongly suggest that lysosomal enzymes. released by the monocytes when incubated with anti-D-sensitized erythroeytes, are responsible for the cytotoxic activity of these cells towards sensitized erythrocytes. The action of these enzymes only occurs over a short range, probably at the site of attachment of the erythrocyte, becuuse only erythrocytes that were bound to the monocytes were lysed. The finding of other investigators that removal of monocytes from suspensions of human mononuclear leucocytes results in a strong reduction in the cytotoxic activity of these leucocytes towards sensitized erythrocytes in vitro. was confirmed.     

Interactions Between Aerolysin, Erythrocytes, and Erythrocyte Membranes

Aerolysin, a hemolytic and lethal exotoxin of Aeromonas hydrophila, was analyzed for amino acids. Assuming 8 histidine residues/mol, the purified toxic protein has, by summation, a molecular weight of 49,000, a value in agreement with earlier estimates by other methods. Erythrocytes from different animal species differ greatly in sensitivity to aerolysin's lytic action. There is some correlation between sensitivity and phosphatidyl choline content. Erythrocyte membranes of different species bind the toxin, and the efficiency of binding is a function of sensitivity to lysis. Binding is temperature independent, is not dependent upon membrane sialic acid, and is decreased by prior treatment with phospholipase C and proteases. Preparations of aerolysin convert substantial amounts of membrane phosphorus to water-soluble form; the conversion is concentration and temperature dependent. Most of the conversion is attributable to contaminating phospholipase(s) that is separable from the toxin. Aerolysin purified by electrophoresis in polyacrylamide gel retains some phospholipase activity, and this activity may or may not be a contaminant.     

Lymphocyte-Associated Complement: Role of C8 in Certain Cell-mediated Lytic Reactions

⁵¹Cr- labelled chicken erythrocytes (E) were treated with human     and C7 to form     , susceptible to lysis by the terminal complement components C8 and C9 ('reactive lysis') Addition of purified and extensively washed human blood lymphocytes, but not of erythrocytes, to     resulted in a similar but cell-mediated reactive lysis. Contamination of the effector cell preparations with plasma was excluded. The reaction does not require living effector cells. Its dependency on C8 was proved by inhibition with rabbit anti-human C8 as well as with its F(ab')₂ fragments. Although terminal complement components may be of importance for cell-mediated lysis of complement-carrying target cell, no effector role could be assigned to them in antibody-dependent lymphocyte mediated lysis Thus, lysis of antibody-coated chicken erythrocytes in the absence of added complement by purified human lymphocytes was not inhibited by the F(ab')₂ fragment of anti-C8.     

Fowl Antibody: V. The Interactions of Fresh and Heated Fowl Serum and of Guinea-Pig Complement Measured by the Lysis of Sensitized Red Cells

Dilutions of fresh fowl serum up to about 1:100 inhibit the lysis of sensitized red cells by guinea-pig complement; this inhibition is decreased by treatments of the serum that also reduce its own haemolytic activity. Studies of this effect are complicated by the fact that fresh fowl serum, when diluted up to 1:32–1:64, lyses sheep red cells with no added haemolysin; with cells sensitized with added haemolysin this lysis is seen in even higher dilutions. Lysis by fowl complement is in turn inhibited by guinea-pig complement: the lytic action of 1–2 C′H₅₀ of fowl C′ is blocked by guinea-pig serum 1:1000. Thus mixtures of sheep red cells sensitized with a mammalian haemolysin, fresh fowl serum and guinea-pig complement possess two distinct haemolytic activities which are mutually inhibitory, giving rise to effects that simulate specific fixation.

Heated fowl serum, if present in excess, inhibits lysis by fowl C′; when added in small amounts it supplies a limiting heat stable factor and enhances lysis.

     

The Conglutination Phenomenon: XII. Immuno-Conglutinin in Experimental Infections of Laboratory Animals

Immuno-conglutinin has been stimulated in rabbits after infection with Listeria monocytogenes, Salmonella typhi-murium and other bacterial species. Infection of rabbits with Western Equine Encephalomyelitis, Hammon Reeves, Murray Valley Encephalitis and Aedes trivittatus viruses also stimulated the production of this substance. The most marked immuno-conglutinin response followed infection of rabbits with Trypanosma brucei.

Rickettsia burneti infection in guinea pigs produced a biphasic immuno-conglutinin response, the first phase being coincident with the rise of antibodies to the Phase II antigen and the second phase with the retarded rise of antibodies to the Phase I antigen.

In mice also, the immuno-conglutinin level was raised following infection with Escherichia coli, Salmonella typhi-murium and other bacterial species.

     

The Relationship Between Cardiovascular and Catecholamine Reactions to Laboratory and Real-Life Stress

Studies on the predictive value of stress testing in the laboratory for the reaction to real-life stress have shown equivocal results. The variety of laboratory tasks differ for unknown reasons in their predictive power, and the results vary unsystematically among the physiological parameters measured. Most studies have focused on the prediction of ambulatory levels of blood pressure. Many other influences, besides stress, however, influence ambulatory levels. Therefore, a better operationalization of real-life stress is to measure a person in a resting position during a period of well defined real-life stress. The present study investigates whether the difference in ability of laboratory stress tasks to predict real-life stress values is due to the different type of physiological response they induce. Hence, in the present study a more detailed measurement of the stress response was performed. A second question was whether stress testing would add to the prediction of the real-life stress reactions above the prediction based on resting levels. This question was answered for both cardiovascular and catecholamine reactions to laboratory tasks. Two active coping tasks, one inducing a mainly cardiac-sympathetic reaction and the other a relatively more vascular response, and a cold pressor test were administered to 33 healthy young males. Real-life stress consisted of the anticipation of the public defence of the PhD thesis. Tasks indeed differed in predictive power, but this was not a function of the type of response they induced.(ABSTRACT TRUNCATED AT 250 WORDS)     

Complement: Mechanisms and Functions

Among glass-adherent peritoneal exudate cells (gaPEC), induced by an inoculation of 1% glycugen solution, about 4.5% were classified as antibody-forming cell precursors (AFCP) on the first day of culture by means of anti-muse B-cell antibody (anti-B-Ab). They proliferated and differentiated into IgG-foming plasma cells when cultured with antigen and thymic RNA in vitro. Pretreatment of PEC with anti-B-Ab and complement suppressed the formation of plasma cells. AFCP had receptors for IgM-antigen complexes and for complement, both of which were independent of Ca⁺⁺ and mg⁺⁺ and resistant to treatment by pronase or phos-pholipase C. Cells bearing detectable receptors for EA(IgM) ad EAC diminished by the 6th day when gaPEC were cultured with thymic RNA, but persisted longer in cultures without thymic RNA. The same percentages of cells demonstrated tartrate-resistant acid phosphatase activities but were devoid of esterase. Twenty to thirty percent of anti-B-Ab sensitive cells ingested latex particles. The proliferation kinetics of IgG-forming cells were studied through the 21st day of culture by means of peroxidase-labeled antibody staining methods.