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Fifty children with idiopathic membranoproliferative glomerulonephritis (MPGN), aged 2-14 years at apparent onset, were monitored for the presence of C3 nephritic factor (C3 NeF) and signs of complement activation in serum. In addition, C3 allotyping was performed in 32 patients. Observation time ranged from 2 to 20 (median 11) years. C3 NeF activity was detected at least once in 60% of the patients (in 11 of 26 with type I, in 15 of 17 with type II, and in four of seven with type III). C3 NeF-positive patients had significantly reduced levels of CH50 and C3 and elevated levels of C3dg/C3d. During follow-up, C3 levels were persistently normal in 62% of the patients with MPGN type I and in 43% with type III but in only 18% with type II. C3 allotype frequencies differed from those found in healthy controls with a significant shift to the C3F/C3FS variants in C3 NeF-positive patients. C3b(Bb)P as a marker for alternative pathway activation was not increased in C3 NeF-positive patients. Despite the presence of C3 NeF activity, C3 levels remained normal in six patients throughout the observation period. C3 NeF became undetectable in six patients, whereas seven developed C3 NeF activity during follow-up. There was no significant difference in renal survival probability in patients with or without C3 NeF activity. Neither C3 variants nor continuous low C3 or low CH50 levels had any prognostic value for the clinical outcome. No factor H deficiency was detected.

61 patients suffering from intrinsic (idiotypic) or extrinsic (allergic) asthma were investigated for signs of complement activation and for C3 phenotype distribution. Activation of both the classical and alternative pathway of the complement system and generation of the membrane attack complex could be assessed by ELISAs for the activation-specific protein-protein complexes C1rsC1 inhibitor, C3b(Bb)P and SC5b-9, respectively. A possible deficiency of the complement regulatory proteins C1 inhibitor, factor H and factor I was excluded. In contrast to earlier studies, C3 allele frequencies did not differ from those found in the healthy population. Our results support the role of complement activation during bronchial asthma and, thereby, provide further evidence for the inflammatory nature of the disease.

Objective To determine the generation of anaphylatoxin C3a in plasma and bronchoalveolar lavage fluid in trauma patients at risk for the adult respiratory distress syndrome (ARDS). Design Prospective study. Setting ICU in a university hospital. Patients Severely traumatized patients at risk for the ARDS (n = 25). Intervention EDTA plasma samples and bronchoalveolar lavage fluid were obtained. Measurements and main results Complement proteins C3, C4, C5, and the inhibitors C1-inhibitor, Factor H, and Factor I were quantitated in EDTA-plasma samples obtained every 6 hrs during the first 48 hrs after ICU admission and every morning from days 4 to 14 after injury. In bronchoalveolar lavage fluid, the complement activation production of C3a-desArg was quantitated and the volume of epithelial lining fluid was calculated. All patients showed a decrease of the complement proteins C3, C4, C5 and of the inhibitors C1-inhibitor, Factor H, and Factor I during the first 24 hrs, indicating complement consumption. Patients developing ARDS (n = 11) showed significantly higher C3 concentrations and a higher C3a/C3 ratio in the first few hours after multitrauma. Follow-up bronchoalveolar lavages demonstrated highly increased amounts of C3a in epithelial lining fluid during the first 24 hrs, mainly in ARDS patients and, to a lesser degree, in non-ARDS patients. To determine the origin of C3a in bronchoalveolar lavages, the ratio of C3a in epithelial lining fluid and plasma was calculated. Conclusion The C3a of epithelial lining fluid to plasma ratio was extremely high in patients developing ARDS, but even the non-ARDS group had a ratio greater than 1, indicating that a substantial local complement activation occurs in the lung.

SUMMARY In vivo and in vitro studies have shown that complement activation plays an important role in the pathogenesis of the adult respiratory distress syndrome (ARDS). In a prospective study of polytrauma patients at risk of ARDS (n= 38) complement parameters were determined over a period of 14 days in serial plasma samples (obtained every 6 h during the first 48 h). Polytrauma induced a rapid and remarkable complement activation. Low levels of the complement proteins C3, C4, C1 inhibitor (Cl INH) factor I and factor H during the first 48 h indicated complement consumption in all patients. Elevated C3a levels in the first few hours after injury were associated with the later development of ARDS. A more sensitive indicator than C3a alone was the calculated C3a:C3 ratio discriminating ARDS and non-ARDS patients. A second rise of C3a levels and C3a:C3 ratio from day 4 on paralleled the course of extravascular lung water. To assess the mode of complement activation, the activation-specific protein complexes Cl rCls-Cl INH and C3b(Bb)P were measured in some of the patients. We demonstrate that in the first 48 h complement activation occurred via the alternative pathway only and was later followed by an additional activation via the classical pathway. Our observations suggest that monitoring of C3a and C3 in plasma can identify polytrauma patients at high risk for ARDS at an early stage of the disease.

Among ten different allergens, house-dust extract proved to be the most potent complement activator. It was therefore chosen to investigate the susceptibility of complement in the serum and bronchoalveolar lavage fluid of patients with extrinsic asthma and control persons. Complement activation in serum was assessed by the appearance of C3d as well as the activation-specific protein-protein complexes C1rs-C1inhibitor (classical pathway) and C3b(Bb)P (alternative pathway). Complement was activated via both the classical and the alternative pathway in a dose- and time-dependent manner. In contrast to earlier observations, however, complement was less affected in the serum of asthmatics than in the serum of normal individuals. Differences were restricted to alternative-pathway activation, probably due to preactivation and/or a significantly higher serum concentration of the regulatory protein factor H in asthmatic patients. In vitro generation of C3a in bronchoalveolar lavage fluid could not be achieved, although the presence of alternative pathway proteins C3, B and D was demonstrated.

The adult respiratory distress syndrome (ARDS) is a well-known complication of traumatic and septic shock. Although other different triggering events cause the development of ARDS, its pathology is surprisingly consistent. It is characterized by intestinal and alveolar edema, hypoxemia due to the intrapulmonary shunting of blood, decreased pulmonary compliance, and increased microvascular permeability in the lungs.

The interaction of d.l. activity with chicken red cells (CE) generates a cell intermediate with the properties of classical E*. Generation of CE* by d.l. activity at 37°C is rapid, while there is a considerable lag in the conversion of CE* to ghost and hemoglobin. Conversion of CE* to ghosts can be blocked by high concentration of EDTA and/or 0°C. CE* contain at least C6 and C9 on their surface.

Sera of patients infected withSchistosoma japonicum consistently reacted in immunoblots with 31 kD proteins fromSchistosoma mansoni worms. Control persons did not react. A human antiserum directed specifically against diagnostic 31 kD antigens of S.mansoni recognized components with the same molecular weight inS. japonicum. These cross-reacting 31 kD schistosome antigens may potentially be applied in a purified form for immunodiagnosis of schistosomiasis japonica.

Summary Rabbits with a hereditary absence of a component activity of complement, C′6, were injected with nephrotoxic sheep serum in amounts which produced only the second phase of nephrotoxic serum nephritis and were found to develop disease of equal severity to that which developed in control animals. Similar results were obtained in defective rabbits which possessed circulating anti-C′6 antibodies or with the use of a γ-globulin fraction of nephrotoxic serum devoid of C′6 activity, thus eliminating the possibility that the results were due to trace amounts of C′6.

Cryoglobulin precipitates of the mixed type, containing 19 S IgM and 7 S IgG gamma globulin, were isolated from a patient serum and the interaction with serum C was studied. Loss of C activity in human serum incubated with cryoprecipitate was accompanied by the generation of C3 activity in the cryoglobulin aggregates. Complete dissociation of cryoprecipitation and the interaction of the precipitates with C was achieved at temperatures below 22°C and above 32°C respectively. The normal C titre in fresh serum of a cryoglobulinaemic patient was significantly reduced in vitro by first cooling and then rewarming the serum. The possible pathogenic role of the bithermic C activation in cryoglobulinaemic vascular lesions is discussed.

Summary The mechanism of rabbit serum bactericidal action on S. typhi 0 901 sensitized with immune antibody has been studied. The bactericidal reaction was separated into three subsequent intermediate reaction steps. An intermediate phase sensitive to MgNa2EDTA and to Na3·HEDTA is followed by a phase sensitive to Na3HEDTA alone. A third phase is reactive in the presence of both MgNa2EDTA and Na3·HEDTA and was identified as the reaction of C′3, as defined in the hemolytic system. All details of the bactericidal reaction tested follow the rules derived for C′ in studies on the hemolytic system. The laws valid for C′ in immune hemolysis seem to apply also for serum bactericidal action on sensitized S. typhi.

A strain of rabbits with an inherited complement deficiency was shown to lack the sixth component of the hemolytic complement system. A method was elaborated for the partial purification of this component from normal rabbit serum. Upon injection of partially purified rabbit C'6 into C'6-deficient animals, an antibody was obtained which specifically inhibited the hemolytic activity of C'6. The data suggest that C'6-deficient serum either lacks the C'6 molecule or contains it in a chemically modified and inactive form.

No complement (C) activation was observed when IgA cold agglutinin was reacted with its antigen on RBC. Neither C-consumption (CH50) nor C1 binding (classical pathway) nor conversion of factor B or C3 (alternative pathway) could be detected. In contrast, IgM cold agglutinins under the same conditions did activate the classical pathway. If the IgA was heat aggregated it was able to activate the alternative pathway as evidenced by factor B and C3 conversion. The result is consistent with the absence of in vivo hemolysis in patients with IgA cold agglutinins.

Nach intravasaler Injektion von jodhaltigen Rontgenkontrastmitteln kann es zu uner-wunschten Nebenreaktionen kommen. Hautrotung, Quaddelbildung, Ubelkeit und Er-brechen sind zwar meist von leichterer Natur, doch werden auch schwere Reaktionen wie Urticaria, Asthma und hypotensiver Schock beobachtet.

A glycoprotein from human erythrocyte membranes has been found to inhibit lysis of target cells by the attack-phase components C5-C9 from human complement. The inhibiting molecule was purified and identified as glycophorin A. Thus, glycophorin A may have a regulatory function in the lytic complement attack on isologous cells.

Schistosomula were recovered from the skin of mice following primary infections. On the surface of such "infecting schistosomula", mouse C3 could not be detected by immunofluorescence. Subsequent incubation in vitro with fresh mouse serum led to the effective deposition of mouse C3 on schistosomula only when they were recovered within a few hours but not after one or two days following infection. In vitro deposited murine C3c was lost from i.v. injected schistosomula in the mouse circulation within one day as was human C3c. Infecting schistosomula exhibited a close to complete resistance to the lytic in vitro activity of human complement. This resistance was complete in older parasites. It existed in spite of the presence of parasite-bound human C9, which was detectable on all developmental stages of schistosomes following incubation in fresh, but not inactivated human serum. Lung schistosomula, 3-week and 6-week-old schistosomes were resistant to cellular cytotoxicity upon incubation with fresh human serum and rat peritoneal exudate cells although cell adherence mediated by human C3b was demonstrated with lung worms. The data suggest that schistosomula may evade in vivo the lytic activity of complement and also complement-mediated cellular cytotoxicity. Depending on the species of serum, this can be demonstrated in vitro by lack of opsonization or by resistance to lytic and cellular attack mechanisms.

Purified human C5a elicits a fast chemiluminescence (CL) response from isolated human granulocytes in the presence of Lucigenin (bis-N-methylacridinium nitrate). The reaction is inhibitable to more than 90% by superoxide dismutase (SOD) - final concentration 200 micrograms/ml -, to about 60% by catalase - final concentration 10 mg/ml - and to 30% by the hydroxyl radical scavenger D-mannit - final concentration 100 mM. Therefore O2- seems to be the oxygen radical responsible for most of the CL, while OH and H2O2 are also involved. Addition of normal pool serum to the cells for 1-2 min before stimulation with C5a strongly enhances the effect in a dose and time-dependent manner. Therefore the existence of a "helper activity" in serum amplifying the C5a-induced CL of granulocytes is postulated. This "helper activity" is, however, no specific for C5a, since CL responses elicited with the chemotactic peptide f-met-phe or by phorbol-myristate-acetate (PMA) are also enhanced by preincubation with serum. In contrast, ConA-induced CL is not enhanced but decreased. Therefore, though not unique to C5a-induced CL, the "helper activity" seems not to represent a general "adjuvans" effect of serum on the granulocytes, but to be restricted to certain stimuli.

In the course of our studies on the structural change of C5 by acidification (U. Rother et al., 1978), we found that the C5 preparations purified according to published methods contained more or less activated C56. When added to sensitive target cells (guinea pig or chicken erythrocytes), C5 mediated lysis by C7-C9 without the addition of C6 or any activation procedure. Generation of C56 was probably due to drastic changes in the physicochemical environment during purification. Such changes like high or low pH or high ionic strength were shown to cause activation. A method for purification of C5 is described in which polyethyleneglycol (PEG) or (NH4)2SO4 precipitation, as well as low or high pH, was avoided. As a last step, traces of C6 were removed by affinity chromatography. The resulting preparation was free of C56. Activation by acidification was not possible without the addition of C6. The total recovery of C5 was 12% with almost no loss of specific activity.

lodinated radiographic contrast media such as methylglucamine diatrizoate and sodium ioglycamate activate serum complement in vitro. This was shown by a dose-, time-, and temperature-dependent decrease of total hemolytic complement activity in normal human serum, consumption of C4 and C6 activity, conversion of C3 to C3b, and generation of C5-derived chemotactic and smooth muscle contracting activity. Complement activation was achieved even in sera depleted of immunoglobulin and properdin, which may indicate that contrast media induce complement activation by mechanisms different from the classical or alternative pathway.

Cellulosic membranes have been shown to activate C3 via the alternative pathway. Generation of the stable product C3d was compared in polycarbonate (PC) and cuprammonium (CU) hollow-fiber minidialyzers with identical geometry, using an in vitro recirculation system with platelet-poor plasma (recirculation rate 20 ml/min; 1,600 fibers; 0.115 m2; 40 ml system priming volume; recirculation time 120 min). After extensive washing, residual protein was removed by reverse transmembrane pressure (1.5 bar) from the air-filled dialysate compartment. Such protein fraction, functionally defined as 'secondary membrane', was similar in quantity for PC and CU fibers. Plasma proteins were present in the membrane without selective adsorption (flat-bed polyacrylamide gel electrophoresis and rocket immunoelectrophoresis using monospecific antibodies). C3d, measured by immunoelectrophoresis, increased with time in the fluid phase compartment of both dialysers, but C3d generation was consistently and significantly (p less than 0.05) less in PC than in CU dialysers. The observation documents an activation of C3 with PC, in contrast to previous reports of no activation. However, C3 activation with PC was less intense than with CU.

Erythrocytes from paroxysmal nocturnal hemoglobinuria patients (PNH-E) are much more susceptible to lysis by acid-activated human serum than normal human erythrocytes. Acidification of normal human serum to pH 6.4 in the absence of erythrocytes generates this lytic activity independently of the alternative pathway of complement activation. A shift of pH of a mixture of purified human C5 and C6 to 6.4 at 0°C generates a similar activity C(56)a that lyses PNH-E together with C7-C9 much more efficiently than normal erythrocytes. Since acidactivation of normal human serum occurs in the absence of C3, the acid-activated C56 appears to be the lytic principle in acidified human serum.

SummaryThe complement activity was determined in sera of recipients after reciprocal skin transplantation between two isogenic strains of BN and Lewis rats. Small or massive allografts did not significantly affect the pattern or the level of overall complement activity, despite the fact that these animal sera are known to bind complement in the cy to toxic reaction in vitro. A slight initial rise of the serum complement activity has been noted in most of the animals. This rise is attributed to a possible increase in production of serum protein elicited by bleeding. The results have been interpreted to rule out either an exhaustion or an inactivation of the complement system as a causative factor in the longevity of massive grafts.

SummaryThree rabbits with an hereditary defect of the classical third component of complement (absence of C′6) were intravenously injected with 3 × 109 heat-killed S. typhi 0 901 labeled with I131. The rates of blood clearance by the RES in these animals were found to be identical with the rates observed in normal rabbits. Ten minutes after the intravenous injection, 84% of the bacteria were found localized in the liver. It is concluded that the activity of C′6 is without influence on the removal of S. typhi from the blood stream by the RES. The nonparticipation of C′6 in the clearance mechanism distinguishes this reaction from the immune bactericidal action of rabbit serum against 5. typhi 0 901.

The term deviated lysis (d.l.) describes a particle bound or fluid phase C function, which lyzes unsensitized cells (E) in the presence of EDTA. Its generation via the classical or the alternative pathway has been described. The activity was stable in the fluid phase. It was found in the region of 180,000–220,000 daltons or 9.7S, respectively, and consisted of C5, C6, C7, C8 and C9. Once generated, the activity could be blocked by the respective antibodies, but not by anti-C3 or anti-B. We now report on a similar activity generated by exposing human serum either to acid pH or low ionic strength. As in C activation by inulin, the factors C5–C9 are involved. Although factor B is converted when normal human serum is brought to pH 6.4, C3 is not. C3, factor B, or properdin, respectively, are not needed. The activation takes place at 0°C within seconds.