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Factors in normal human serum that are required for opsonophagocytosis and intracellular killing of Pseudomonas aeruginosa by human polymorphonuclear leukocytes were investigated. IgG natural antibodies, properdin, and the proactivator of the third component of complement were found to be essential to these events. The addition of immune IgG antibodies to the system eliminated the requirement for properdin and the proactivator. These observations suggest that two mechanisms of complement activation can be involved in opsonophagocytosis of P. aeruginosa.

Seven patients with dermatitis herpetiformis (DH) were studied employing immunofluorescent techniques with monospecific antisera to various immunoglobulins, complement, and alternate pathway components. This study demonstrates: 1. 1. IgA deposition occurs adjacent to the skin basement membrane in a granular pattern and is unrelated to disease activity. 2. 2. C3 proactivator (C3PA) was found in areas corresponding to IgA deposition in four patients and a similar properdin deposition was found in one. 3. 3. C3 in sites corresponding to IgA deposition was found only in active disease. No C3 deposition was found in patients whose disease was controlled with dapsone. 4. 4. Clq was found in only one of 20 biopsies. No definite C4 staining was found. These observations are consistent with the suggestion that complement activation, mediated by IgA via the alternate pathway, is involved in the pathogenesis of the skin lesions in DH.

Human properdin contains 9.8% carbohydrate consisting of 3.8% hexose, 0.7% fucose, 1.5% hexosamine and 3.8% sialic acid. The partial specific volume for properdin calculated from amino acid composition and carbohydrate analysis was 0.7 cc/g. The mol. wt determined by high speed equilibrium ultra-centrifugation was 184,000 ± 12,000. Results from SDS-polyacrylamide gel electrophoresis and SDS or guanidine agarose chromatography provide consistent evidence that properdin is comprised of four non-covalently linked subunits of similar mol. wt (46,000). The subunits combine in aqueous media to form a dimer (4.6 S, mol. wt = 88,000. On equal weight basis, the dimer has about 70–85 per cent of the antigenic properties and half the hemolytic activity of the native protein.

Properdin (P), a highly basic euglobulin, was purified from human serum to molecular homogeneity without the use of zymosan. Isolated P was found to efficiently initiate activation of the alternate pathway of complement activation (C3 activator or properdin system) and to be an essential component during its early reaction stages. The activity of isolated P did not require the presence of an activating polysaccharide. It was therefore concluded that purified P had been obtained in an activated form (P). In an isolated reaction system containing purified C3, C3 proactivator (C3PA), and C3 proactivator convertase (C3PAse), P was able to mediate the activation of C3PAse which in turn activated C3PA to cleave C3. This activation of C3PAse was found to depend on the presence of native C3. These results allowed the formulation of a concept in which P is envisaged to act as a modulator of native C3 enabling it to activate C3PAse. Activation of C3 was efficiently mediated by P in the fluid phase. Efficient activation of C5, however, required the participation of an insoluble polysaccharide (zymosan). The possibility is raised therefore that P might also be an integral part of the multimolecular C5 convertase of the alternate pathway of complement activation.

The protein in the properdin pathway responsible for conversion of precursor factor D to D has been isolated and found to be identical with properdin. Sequential ion exchange and gel filtration chromatography demonstrated identity between properdin protein, measured by radial immunodiffusion, and the capacity to activate D to D, assessed by formation of the intermediate, EAC43B(D). Properdin, purified in this manner, was homogeneous on acid polyacrylamide disc gel electrophoretic analysis, with the band of protein corresponding to the position of eluates in the replicate gel capable of activating highly purified D. Demonstration of the homogeneity of purified D by alkaline disc gel electrophoresis, coupled with the linear stoichiometric hemolytic titrations of each factor, indicates that direct interaction between properdin and D generates D. Thus, activation of D by properdin represents a mechanism in the properdin pathway by which D becomes available for formation of the C3b-dependent C3 convertase.

Four patients who developed symptoms of Goodpasture’s syndrome during the winter 1971–1972 from a geographic area of about a 25mile radius have been studied. There was an epidemiological association b

SUMMARY A fibrin slide technique was utilized to study the relationship between renal homograft rejection and glomerular plasminogen activator activity. Glomerular plasminogen activator activity was absent in tissue from 25 of 30 patients rejecting kidneys within 10 months of transplantation. On the other hand, glomerular activator activity was detected in tissue from patients with acute tubular necrosis after transplantation, in functioning grafts, in grafts rejected more than 2 years after transplantation, and in tissue from 328 patients with a variety of renal diseases. These studies suggest that loss of glomerular plasminogen activator activity may be a diagnostic feature of renal homograft rejection occurring within 10 months of transplantation.

C3 and C4 serum complement component levels were examined during various stages of disease activity in 14 SLE patients. It was found that C3 levels were more sensitive index of disease activity than those of C4. C3 levels lower than C4 as expressed by a C3/C4

Evidence is presented that phenomena ascribed to the properdin system may be explained in terms of classical antibody (Ab) in combination with three of the components of complement (C'), i.e., C'1, C'4, and C'2 respectively. The results suggest that the complex of properdin (P) with zymosan (Z) represents a mixture of Z·Ab, Z·Ab·C'1, 4, Z·Ab-C'1, 4, 2, and Z·Ab·C'1, 4, 2 in a decayed state. The purported preferential reactivity of Z with the third component of C' (C'3) is not supported by the present experiments in which Z was reacted with C' in both guinea pig and human sera. Approximately the same number of reactive units of C'1, 4 and of C'3 were inactivated by Z, as well as by D. pneumoniae and a washed specific precipitate of bovine albumin-anti-albumin. The latter evidence that small amounts of antigen-antibody complex fix significant amounts of C'3 stands in contrast with the classical concept that C'3 is not fixed in ordinary complement fixation reaction. The observed reactivity of C'3 is explained on the basis of the present use of essentially undiluted serum as a source of C'3 and of 37° as the temperature for fixation. Ancillary data indicate that purified properdin contains Ab. In the presence of a chelating agent and at 0° properdin agglutinated Z granules. Measurements of nitrogen (N) uptake by Z suggest that 0.25 µg. N were contributed by solutions stated to contain 1 unit of properdin. The broad spectrum of reactivity or cross-reactivity of the Ab in normal serum is likely due to the wide distribution in nature of closely related polysaccharides. Further immunochemical studies are necessary to establish definitively the origin and mode of action in "natural resistance" of antibodies reactive with these polysaccharides.

The purpose of the present investigation was to clarify the nature of normal antibody responsible for the bactericidal activity of normal serum mainly against Gram-negative bacilli, as comparing with properdin, a new bactericidal entity in serum, reported by Pillemer et al.Properdin-zymosan or normal antibody-organism complexes were respectively prepared by the absorption of normal serum with zymosan, Sh. dysenteriae 1 and Sal. typhosa 0-901 W at the defined temperatures. These complexes were placed in the reservoirs of agar gel in a barbital buffer, pH 8.2, ionic strength 0.05, and then the immunoelectrophoresis was performed. The obtained results were as follows.1. Both of the normal antibody and properdin of human serum were demonstrated as a single precipitin arcs at β1-globulin position; those of rabbit serum at α2, p1-globulin position. It was, furthermore, shown that normal antibody is substantially different from properdin.2. By means of the mutual absorption of rabbit serum with Sal. typhosa and Sh. dysenteriae, the precipitin arcs of the normal antibodies against these bacteria were individually observed at the same position of α2, β1-globulin. Accordingly, it may be taken to indicate that normal antibodies against enteric bacteria posses a marked specifity, in contrast with properdin which is non specific.3. The precipitin arcs of the immune antibodies against zymosan and Sh. dysenteriae, on the other hand, were clearly observed at the γ-globulin position. The above-mentioned findings suggest that normal antibody and properdin, in substance, differ from so-called immune antibody.

Summary A study has been made of the functional replacement of Mg++ by other cations in the interactions of properdin and complement with bacteria and with zymosan. Of 22 cations tested, only Mg++, Co++ and Mn++ were able to restore bactericidal activity to serum that had been treated with cation-exchange resin. Mg++ was quantitatively the most effective, being twice as active as Co++ and five times as active as Mn++ on a molar basis. Ca++ was completely inactive. Mg++, Co++ and Mn++, when added to resintreated serum at levels that effectively restored bactericidal activity, also restored the ability of the serum to form a properdin-zymosan complex with zymosan at 17°. However, only Mg++ and Co++ restored to resin-treated serum the property of rendering C′3 susceptible to inactivation by zymosan at 37°C. These findings point out differences between the mechanism of the bactericidal activity of the properdin system and the inactivation of C′3 by zymosan.

SummaryMethods have been presented for the preparation of properdin from resin-collected and from ACD plasma by the application of the zymosan adsorption technic of Pillemer et al. Preliminary data are presented suggesting the feasibility of the isolation of properdin from Cohn Fraction I without the use of zymosan.

Incubation of human serum with zymosan at 37°C, followed by removal of the zymosan residue by centrifugation, resulted in C3-zymosan complex. The C3-zymosan complex could be dissociated and C3 recovered in the eluate. Preparations of dissociated C3 did not contain the hemolytically active component of complement, C1 esterase, or fibrinolytic or caseinolytic activity. Addition of dissociated C3 to EAC142 blocked the formation of EAC1423 or EAC14235 by partially purified C3,C5. The effect of the inhibitor was not limited to intermediate complexes, it also blocked the hemolytic activity of serum was restored by adding C2, but not C3. A positive immune adherence test was obtained by treating EAC142 with dissociated C3. The data are discussed in relation to an hypothesis that the inhibitor in preparations of dissociated C3 is C3, which is hemolytically inactive, but participates in immune adherence and has greater affinity for SAC2 than the C3 molecule possessing functional integrity.

The properdin system is a serologic protective system which contribute to natural resistance and does not belong to cellular protective system.The properdin titers were measured in order to explain the significance on every pathologic form of chronic tonsillitis, especially on the influence of properdin upon the mechanism of tonsillar focal infection.The method of the Zymosan assay modified by McNall was used.Average values of the serum properdin titer in the patients with focal chronic tonsillitis showed 77.1 units. These were lower than the 89.3 units in the patients with simple chronic tonsillitis and the 90.4 units in a control group of healthy persons. Those with rheumatic disease especially showed 74.6 units which was the lowest among the systemic diseases.In a focal group low values of the serum properdin (under the lowest of the serem properdin titers in a control group, 60 units) were seen in 31.8%, while 9.5% in a simple group.Low values in serum properdin were found more frequently in patients who showed an increase in Antistreptolysin-O titer and the properdin values in focal group were lower than those in the simple group.This is supposed that streptococcal infection gives the impeded effect to individual resistance.After focal elimination by tonsillectomy, the serum properdin remained much the same value as before tonsillectomy in both simple group and non-focal disease with chronic tonsillitis. But in the focal group, the serum properdin increased close to the nomal level and its value was in parallel with the degree of the recovery from systemic disease.Results from cutaneous injection made with the diagnosis solution of Paspat, focal group showed frequently an increase of serum properdin in coopration with reduced erythemata after tonsillectomy, while the simple group did not show these variations due to removal of tonsil.This increase of properdin suggests a recovery of natural resistance, because the patients became to possess no allergic aggresion on basis of bacterial infection.The properdin titers were varied in tonsillar nephritis, after tonsillar direct irradiation by ultrashort wave was given, and the titer in tonsillar rheumatic disease increased, after tonsillar focus was annuled by Impletol injection.From these results, it was attested that the tonsillar focus had the influence upon natural immunity and suggested that the properdin system had some roles in the mechanism of the tonsillar focal infection. Therefore the measurement of the properdin value has the effects to infer the immune stadium in the basis of the focal infection.

A new method of properdin determination in human serum is presented. The method of immunoelectrophoresis described by Afonso was adapted to quantitative measurement of serum properdin level. The ring diameter of immunodiffusion on agar plates was determined as a measure of the concentration of properdin protein. Simultaneously, in every sample the properdin nitrogen was measured according to Kogler and co-workers. The correlation between the ring diameter and serum properdin nitrogen level is statistically adequate. The new method of using immunodiffusion for the quantitative determination of properdin may be useful in practical laboratory work.

An Hand von 19 Fallen wird das Verhalten von Properdin, Komplement (C′) und der C′3-Komponente des Komplements nach typischen gynakologischen Operationen mit unterschiedlichem Gewebstrauma studiert. Dabei last sich folgendes feststellen: 1. Bei der posttraumatischen Entzundung sinkt der Properdinspiegel relativ rasch ab, verharrt einige Tage auf niederen Werten und steigt bis zum 10., spatestens 15. Tag wieder auf die Ausgangswerte. Das Komplement (C′) zeigt keine deutlichen Veranderungen. Die 3. Komplementkomponente (C′3) fallt am 1. Tag ab und steigt bis zum 3. Tag uber den Ausgangswert wieder an. 2. Beim Vergleich zwischen den Verlaufen nach Totalexstirpationen des Uterus als schwere Traumen und den Scheidenplastiken als leichte Traumen finden sich keine eindeutigen Unterschiede (im Gegensatz zu fruher beobachteten Serumeiweisveranderungen). 3. Die Aufschlusselung der untersuchten Falle im Hinblick auf die angewandte Thrombose-Prophylaxe mit einem Cumarinderivat ergibt keine deutlichen Unterschiede gegenuber den nichtbehandelten Patientinnen. 4. Das Verhalten von Properdin und α2-Globulinen bei posttraumatischer Entzundung ist zwar gegensinnig, aber zeitlich eindeutig gegeneinander verschoben, so das eine gegenseitige Beeinflussung in vivo schwer vorstellbar ist. 5. Auf die moglichen Zusammenhange zwischen Properdinbindung durch Polysaccharide, Cytolyse, Gewebszerfallsgrose und α2-Globulinvermehrung wird hingewiesen.

80 rats were given zymosan at different doses. The influence on the development and evolution of 3,4-benzpyrene tumour was studied. Small doses (1 mg/kg) of zymosan, administered before the carcinogenic treatment or even after the tumour occurrence, were found to prolonge the latency period of the neoplasm and the animals survival in comparison with animals treated with the carcinogenic hydrocarbon only. On the other hand, high doses (10 mg/kg) administered either before or after the tumour occurrence, were observed to induce a much more rapid development of the tumour and a diminution of the survival time, together with a more sensible decrease in weight.

An analysis of the serum factors involved in the lysis and agglutination of artificially altered red cells in compatible human serum has been presented. Human sera have been found to vary widely in their capacity to hemolyze a panel of artificially altered red cells. These differences have been shown to be the result of a multiplicity of more or less specific serum factors for each altered red cell type. These factors have many of the properties commonly associated with classical antibody. These serum factors are different from complement and properdin. The range of specificity and the degree of cross reaction for these various serum factors have been analyzed. These serum factors are important in the consideration of the use of enzyme treated red cells for the detection of incomplete antibodies, and of certain acquired hemolytic states in man.