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Methods for the preparation and standardization of reagents suitable for studies on the bactericidal action of the properdin system are described. The preparation and properties of serum free of properdin (RP(b)) are presented in detail because of the necessity for a suitable RP(b) in these studies. The properdin system is responsible for the bactericidal action of normal human serum against a variety of microorganisms. The present work shows that the removal of properdin from serum also removes bactericidal activity. Addition of properdin to properdin-deficient serum restores bactericidal activity. A quantitative relationship exists between the final properdin concentration and bactericidal activity against sensitive organisms. The possibilities of a bactericidal assay for properdin are discussed. It is demonstrated that, in addition to properdin, the four components of complement (present in RP(b)) are necessary for the destruction of properdinsensitive bacteria. If any component is missing, bactericidal activity is lost; when the component is replaced, bactericidal activity is restored. Magnesium is also necessary for the bactericidal activity of the properdin system. Maximal bactericidal activity is obtained with magnesium concentrations similar to that of normal human serum (10(-3) to 10(-4)M). The bactericidal activity of the properdin system occurs only at temperatures above 15 degrees . Resistant strains have been encountered in species of bacteria sensitive to the properdin system. Resistance or sensitivity is a characteristic of the individual strain and not of the species. The widespread occurrence of the properdin system in normal mammalian serum and the variety of bacteria destroyed by it suggest that the properdin system is a factor in natural resistance.

Detailed experiments are presented which indicate that the properdin system is an inhibitor of Newcastle disease virus. Viral inhibition required all known components of the properdin system: properdin, all four components of complement and magnesium; the removal of any one constituent resulted in a loss of inhibition; the replacement of the constituent restored antiviral effect. The inhibition of virus was temperature-dependent. The process of inhibition by serum resulted in a decrease in the amount of properdin available in the serum without any measurable effect on the components of complement. The prolonged incubation of inactive serum-virus mixtures with cation-exchange resin resulted in the restoration of some, but not all, of the hemagglutinating activity of the virus. The requirements of the properdin system and the implication of these findings were discussed.

Human complement is inactivated by plasmin, the proteolytic enzyme of plasma or serum active at or near neutrality. The addition of streptokinase to human serum, which converts plasminogen to plasmin, also causes the inactivation of complement components C'2 and C'4 and varying amounts of C'1. C'3 is the most resistant to inactivation by plasmin. Chloroform-activated human plasmin and bovine plasmin also destroy these components of complement, but are less effective than the streptokinase-activated enzyme. The inactivation of complement by the addition of streptokinase to human serum is inhibited by high hydrogen ion concentrations, low temperature, and elevated ionic strength. The inactivation of the components of complement in various fractions of serum is influenced by the available plasminogen and the content of plasmin inhibitors in these fractions. Certain similarities are pointed out between the components of complement and the factors in the plasmin system and between the inactivation of the components of complement by antigen-antibody reactions, by specific agents, and by plasmin. The possible significance of these relationships in immune hemolysis and complement fixation, and the possible role of the plasmin system in the instability of complement and the development of anticomplementary properties in serum are discussed.

It has been shown that injection of lipopolysaccharides, derived from a variety of Gram-negative bacterial species, evokes in mice a rapidly developing rise in resistance to infection with Gram-negative pathogens. This is accompanied by an elevation in properdin titer, at times to levels 2 to 3 times the normal. The rate, magnitude, and duration of these responses are dependent on many factors, the most important of which are the quantity and timing of the lipopolysaccharide administered. The increased resistance to infection evoked in mice by lipopolysaccharides was effective against infections produced by endotoxin-bearing organisms-bacterial species highly susceptible in vitro to the bactericidal action of the properdin system. Properdin titers of mice prior to infection provide an incomplete picture of the subsequent reaction of the host to the infective agent. Following infection with Gram-negative organisms, properdin levels accurately reflect the bacteriologic course and outcome of the infection. Thus, in control animals, properdin titers progressively declined and the animals died, while in mice appropriately treated with lipopolysaccharide, properdin levels were either maintained in the normal range or increased, depending on the dose and time of administration of lipopolysaccharide; this was always accompanied by successful management of the infection. The complex nature of the alterations produced in the host by lipopolysaccharides is stressed. It is pointed out that the increase in the ability of the host to cope with Gram-negative infections may be the result of stimulation of other defense mechanisms, in addition to the properdin system.

Methods for the purification and crystallization of tetanal toxin are described. The methods consist of the multiphase fractionation system involving methanol as the organic precipitating agent under controlled conditions of pH, ionic strength, protein concentration, and temperature. Crystalline tetanal toxin has an electrophoretic mobility of 2.8 x 10(-5) in veronal buffer of 0.1 ionic strength at pH 8.6. The solubility of freshly prepared toxin is essentially constant. The isoelectric point is 5.1 +/- 0.1. The crystalline toxin contains 1 per cent sulfur, traces of phosphorus, and gives the usual protein reactions. It does not contain carbohydrate. The crystalline toxin does not precipitate anti-Clostridium tetani rabbit serum. The final product contains between 3400 and 3600 Lf and about 6.6 x 10(7)M.L.D. per mg. N. Crystalline tetanal toxin is spontaneously converted to a flocculating atoxic dimer upon standing at 0 degrees . This change is accompanied by the appearance of another molecular species as judged by constant solubility tests. Ultracentrifugal analysis of these fractions reveals that tetanal toxin has a sedimentation constant of 4.5 Svedberg units while the atoxic flocculating dimer sediments at 7 S.

Ca(++) potentiates the inactivation of human complement by streptokinase-activated plasmin and chloroform-activated bovine plasmin. The optimum concentration of Ca(++) varies between 10(-2)M and 10(-3)M, decreasing with increasing concentrations of streptokinase. The susceptibility of the components of complement to inactivation by plasmin is different in the presence and absence of Ca(++). C'2 and C'4 are most readily inactivated by plasmin in the presence of Ca(++), while C'1 disappears first in the absence of Ca(++). A large part of this C'1 disappearance is due to spontaneous inactivation. Similarities are pointed out between the influence of Ca(++) on the inactivation of the components of complement by antigen-antibody systems and by plasmin.

High molecular weight polysaccharide complexes derived from normal and neoplastic mammalian tissues were found to combine with properdin and to inactivate C'3 in vitro. These polysaccharide preparations were also found to alter properdin levels and non-specific resistance to Gram-negative infection in mice. In these manifestations, the tissue polysaccharides bore a marked resemblance to bacterial lipopolysaccharides. Some implications of the interactions of tissue polysaccharides and properdin in certain disease states are considered.

The administration of a single small dose of bacterial lipopolysaccharide produces in mice a considerable rise in properdin levels. This is accompanied by an early, transient, non-specific increase in resistance to certain bacterial infections. Bacterial lipopolysaccharides were shown to possess far greater activity than other substances previously studied in bringing about an elevation of properdin levels. After the injection of bacterial lipopolysaccharides, high molecular weight substances appear in the circulation, which interfere with the combination of properdin with zymosan and thus affect the assay of properdin. The administration of small amounts of bacterial lipopolysaccharides to mice at appropriate times before experimental infection "conditions" the mice so that they maintain normal or elevated properdin titers during the infectious process in contrast to control mice which show a progressive decline in properdin to low levels and death. The significance of this observation and its relationship to natural resistance to Gram-negative pathogens are considered.

Considerable purification of streptolysin S has been achieved by fractionation of crude bacterial filtrates with methanol, under controlled conditions of pH, ionic strength, and temperature. The final material (P III) consisted of two electrophoretic components. The purified material was employed for kinetic studies, the results of which were confirmed with hemolysin isolated electrophoretically from P III. The heat of activation (temperature characteristic) of streptolysin S was found to be 17,900 calories per mole. Time dilution curves of the hemolysin were found to be sigmoid. The influence of certain ions and lipids on the course of hemolysis was investigated. The significance of the prolonged lag period and of the deviation in the behavior of streptolysin S from Ponder's equation has been discussed.

A detailed method for the zymosan assay of properdin is described, together with procedures for the preparation and standardization of reagents employed in the test. The reliability and reproducibility of the assay, as well as its limitations, are discussed.