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The effect of propylthiouracil (PTU) on the lytic activity of complement in rat serum was investigated in vivo. Rats (180+/-10 g) were treated daily by gavage with PTU doses of 1-50 mg/200 g body weight for time intervals ranging from 1 to 30 days. Serum classical pathway (CP) and alternative pathway (AP) activities were determined 24 h after the last dose. A single dose of 50 mg/200 g body weight was administered to additional groups and the animals were sacrificed after periods of 1-48 h. The results showed a relatively small reduction ( approximately 30%) in CP activity, evident only in animals treated with 50 mg of PTU for three weeks. However, a clear and opposite effect of PTU, an increase in lytic activity reaching values up to 180% of controls, was observed on AP activity. This effect was seen at all PTU doses used, and occurred within 4 days of treatment with the highest dose. Maximum activity was observed at intermediate intervals, depending on the PTU dose, with a return to control levels occurring after the longer periods of treatment. The lytic activity of serum from animals treated with a single PTU dose of 50 mg/200 g body weight and sacrificed 1-48 h after dosing did not differ from controls. Serum levels of thyroid hormone (triiodo L-thyronine, T3, and thyroxine, T4) were determined in representative groups of treated animals (injected with 5 mg of PTU/200 g body weight/day). These were either undetectable or considerably lower than those of controls. The serum PTU levels of these rats increased for up to 22 days, reaching values of 2-4 microg/ml.PTU is described in the literature as a modulator of both cellular immune responses and antibody production. Upon complement activation fragments of complement components bind to immune complexes and to specific receptors on cells of the immune system. Thus, alteration in AP activity caused by PTU treatment suggests a possible mechanism by which the drug exerts its modulatory effect. Increased complement AP activity might affect events as antigen presentation and hence the onset and course of the immune response.

The main alkaloids isopyruthaline (It1), fangchinoline (It2) and isothalictrine (It3), isolated from Isopyrum thalictroides (L.) were investigated in complement-mediated reactions. The alkaloids influenced the alternative pathway (AP) activity in normal human serum (NHS). They enhanced the inhibitory action of complement activators--carrageenan (Car), zymosan (Zy), hydrogen peroxide (HP) and high temperature via classical pathway (CP) in NHS. Substances strongly potentiated the action of zymosan and cobra venom (CV) in guinea pig serum (GPS). It was established that they could provoke C3 conversion in NHS and mouse sera (MS). The antiinflammatory properties of the alkaloids were evaluated in mouse paw oedema induced by CV, Zy and histamine (His). Isopyruthaline and isothalictrine suppressed paw swelling in CV- and Zy-oedema. They were applied in Zy-induced multiple organ dysfunction syndrome (MODS) in mice. The alkaloids inhibited the increase of the serum complement activity provoked by the injection of zymosan. Itl lowered the mortality rate of mice with MODS if its application proceeded Zy. An increase of the number of mice without tissue injury was established after treatment with It1 and It3.

Nocardia lysozyme digest (NLD) was administered intraperitoneally (i.p.) at a dose of 500 micrograms/kg to normal and immunosuppressed mice for 3 consecutive days prior to inoculation with Klebsiella pneumoniae. A protective effect was observed when the pathogen was injected subcutaneously and intravenously, as opposed to an aggravating effect obtained in the case of intraperitoneal inoculation The i.p. administration of NLD partially restored the immunosuppression caused by cyclophosphamide but did not change cobra venom-induced deterioration of the infection. The results obtained could be regarded as a consequence of the lowered alternative pathway serum complement activity and the crucial role of the diminished level of complement in the peritoneal cavity.

Rosmarinic acid (RA), a naturally occurring extract from Melissa officinalis, inhibits several complement-dependent inflammatory processes and may have potential as a therapeutic agent for the control of complement activation in disease. Rosmarinic acid has been reported to have effects on both the classical pathway C3-convertase and on the cobra venom factor-induced, alternative pathway convertase. In order to define the mechanism of inhibition, the effect of RA on classical and alternative pathway lysis, C1q binding, the classical pathway convertase, the alternative pathway convertase, membrane attack pathway lysis and the generation of fragments of C3 and C5 during activation, was tested in vitro. The results showed that RA inhibited lysis by the classical pathway more than by the alternative pathway. This effect was dose-dependent with maximum inhibition of classical pathway lysis observed at 2.6 mmoles of RA. There was little effect on C1q binding or on the classical and alternative pathway convertases. However, there was highly significant inhibition of lysis of pre-formed EA43b cells by dilutions of human or rabbit serum in the presence of RA (1 mM); this was accompanied by inhibition of C5a generation. We conclude that the inhibitory effect of RA involves the C5 convertase. Such inhibition could be advantageous to the host in disorders where the terminal attack sequence plays a role in pathogenesis.

An extraordinary potent anti-complementary substance was isolated from the root of Lithospermum euchromum Royle (Japanese name: Nan-shikon) which activates the complement system in vitro, and the active principle was shown to be acidic polysaccharide (LR-polysaccharide IIa). LR-polysaccharide IIa was purified by chromatographies on DEAE-Sepharose, Sephadex G-100, concanavalin A-Sepharose, Ricinus communis agglutinin conjugated Sepharose, Sepharose CL-6B and Sepharose CL-2B. LR-polysaccharide IIa was found to be composed of rhamnose, fucose, arabinose, xylose, mannose, galactose and glucose in the molar ratios of 2.0:2.5:3.4:2.8:5.6:9.6:14.4. The polysaccharide also contained 15% of galacturonic acid and 3.8% of protein. The methylation analysis of the polysaccharide showed that rhamnose, arabinofuranose, xylose, glucose and galactose are present as a part of the nonreducing terminal residues. The main chain and side chains are composed of ----3Glc1----,----3Gal1----,----6Man1----,----4Gal1, [corrected] and the branching points consist of (formula; see erratum) These results indicated that LR-polysaccharide IIa has a highly complicated structure. A marked consumption of C4 was observed after the incubation of the serum with LR-polysaccharide IIa in the presence of the Ca++ ion. The anti-complementary activity of LR-polysaccharide IIa was reduced partially in the absence of the Ca++ ion. After the incubation of the serum with LR-polysaccharide IIa in the absence of Ca++ ion, a cleavage of C3 in the serum was found to have occurred through immunoelectrophoresis as well as from the consumption of the complement when rabbit erythrocytes were used in the assay system. These results indicate that the mode of complement activation by LR-polysaccharide IIa is via both the alternative and classical pathways. Complement titer also decreased in guinea pigs upon i.p. injection of LR-polysaccharide IIa.

When human serum was incubated at 45 degrees C for 30 min, C3 and B were converted to C3b and Bb. Molecular weights of purified C3 and B were shown not to change after incubation at 50 degrees C. Spectropolarimetry indicated that the secondary structures of C3 and B changed after incubation at higher temperature. The titration of SH groups in the C3 molecule showed the liberation of an SH group. These results show that the alternative complement pathway is activated at raised temperatures without known activators such as zymosan or lipopolysaccharide (LPS). This may be due to the accelerated interaction of conformationally changed components of the alternative pathway such as C3 and B. Using this system, effects of various substances on the interaction of C3 and B in serum and the purified system were investigated. The addition of arginine and lysine resulted in the inhibition of the conversion of C3 and B in the serum at elevated temperature. Other amino acids such as anionic amino-acids and NaCl did not influence the conversion. In the purified system, only arginine and lysine prevented the conversion of C3 and B, when C3, B and D were incubated in the presence of Mg++ and amino-acids. Since lysine and arginine did not inhibit the enzymatic activity of D, these data suggest that arginine and lysine prevent the interaction of C3 and B in the serum at elevated temperatures.

The anti-complementary effects of the surface-active immunological adjuvants dimethyldioctadecylammonium bromide (DDA) and pluronic polyols L101 and L121 were investigated in the mouse system. All three adjuvants showed complement (C)-inactivating effects. DDA caused a time- and dose-dependent reduction of alternative pathway (AP) and overall C activity, which varied with the serum concentration. Polyols induced a preferential inactivation of the AP by a more direct mechanism. A rather general, causative relationship between anti-complementary and immunological adjuvant activities is suggested. This might involve interference with nonspecific elimination of antigen, counteraction of immunosuppression by terminal C components, and/or moderation of C3b-mediated reduction of Ia-expression, leading to a better antigen presentation.

Interference with hemolytic complement activity by polyanionic substances was studied in relation to the ability of these compounds to act as an adjuvant for a dead listeria vaccine. Heparin appeared a poor inhibitor of the mouse alternative pathway not only in contrast to its effects on the mouse classical and the human classical and alternative pathways, but also when compared to two polyanions with known adjuvant activity: dextran sulfate and suramin. For the three polyanions mentioned a correlation between adjuvant activity and mouse alternative pathway inhibition was observed. These findings suggest a possible causal relationship between interference with alternative complement pathway activation and adjuvant activity.