The membrane attack mechanism of complement. Reversible interactions among the five native components in free solution.

Reversible interactions in free solution were demonstrated to occur (a) between C5 and C8, (b) between C5, 6, 7 and C8, and (c) between C8 and C9. No interaction was observed between C8 and C6 or C7 and between C9 and C5, 6, 7. Interactions between C8 and C9 were enhanced at lowered ionic strength (0.05) and a molar excess of C8 over C9. Complex formation was independent of pH over the range of 6.5-8.5. Under optimal conditions the C8, 9 complex had a sedimentation coefficient of 10.2-10.6S, while native C8 and C9 sedimented at 8.5 and 4.8S, respectively. Specificity and reversibility of these interactions were established. In spite of the limited number of interactions observed, all five of the native proteins of the membrane attack mechanism interacted to form an association product that sedimented at 10.8-11.2S. Demonstration of this product in free solution supports the concept that C5-9 on acquisition of cytolytic activity assemble into a stable multimolecular complex.

Molecular analysis of the membrane attack mechanism of complement.

The molecular arrangement of the membrane attack mechanism of complement was explored. The molar ratios of the components within the C5-9 assembly on the target cell surface were determined using human complement proteins in highly purified and radiolabeled form. With the aid of monospecific complement antisera it was possible to probe the spatial relationships between the components of the assembly. C5 and C6, in the presence of C7, were bound to EAC1-3 in equimolar quantities irrespective of the amounts and the relative proportions of C5, C6, and C7 offered. The amount of C8 bound to EAC1-7 increased with input and at saturation of all C8 binding sites the molar ratio of bound C8/bound C5 approached 1.0. Uptake of C9 by EAC1-8 increased with input and at saturation of all C9 binding sites the molar ratio of bound C9/bound C8 became 6.0. However, calculations suggest that the binding of three C9 molecules to one C8 molecule is sufficient to achieve a full hemolytic effect. Evidence was obtained indicating that binding and hemolytic function of C9 depends upon cooperative interaction of multiple C9 molecules. Binding of C8 to EAC1-7 and the generation of hemolytic C8 sites were inhibited by antibody to either C5, C6, or C7. Uptake of C9 by EAC1-8 and the generation of hemolytic C9 sites were strongly inhibited by anti-C8 and to a lesser degree by anti-C5. Binding of C9 (but not hemolysis) was also reduced by antibody to C6 or C7. The data are consistent with the concept that the fully assembled membrane attack mechanism of complement consists of a decamolecular complex: a trimolecular arrangement composed of C5, C6, and C7 forms the binding site for one C8 molecule which in turn furnishes binding sites for six C9 molecules, saturation of three sites apparently being sufficient for expression of full cytolytic activity of the complex. This work made it possible to design a simple molecular model.

Detection of complement activation by counterimmunoelectrophoresis (CIE).

Counterimmunoelectrophoresis (CIE) was used as a method of detecting activation of the third component of the complement system (C3). Highly purified C3, normal human serum (NHS), EDTA-treated plasma and serum activated with aggregated human immunoglobulin (agg-IgG) or inulin were used as sources of C3 and/or C3 split products. Activation of the alternative pathway of complement was assayed in the presence of EGTA (10 mM) and MgCl2 (0.3 mM), conditions which block activation of the classical pathway. When purified native C3, fresh NHS and fresh EDTA-plasma were tested in CIE against either antisera to whole C3 or to C3 split products, only one precipitin line was found, which was identified as native C3. However, when serum activated with agg-IgG or inulin were tested against the same reagents, two precipitin lines were seen. The first, with more cathodal mobility was identical to that of native C3. The second line had a more anodal mobility, was distinctly separated from the first and contained C3c and C3d as shown immunochemically with specific antisera. Native C3 and split products of C3 were identified by this CIE method in patients showing evidence of activated complement by having subnormal total complement (CH50) levels. When C3 split products were identified, the C3c-C3d precipitin line could always be distinguished from native C3 by its different electrophoretic mobility, even when C3 concentrations in serum varied from 0.25 mg/ml to 1.5 mg/ml. The sensitivity of CIE was compared to that of CH50 by asssaying at different time intervals after agg-IgG was added to fresh NHS. C3c-C3d split products were detected by CIE before any fall in CH50 and at all times when a significant decrease in CH50 was present. This study shows that the CIE technique is a highly sensitive, specific and rapid method for detecting activation of the complement system via classical or alternative pathways in human disease.

Cryoprecipitates in Kawasaki syndrome: association with coronary artery aneurysms.

Cryoprecipitates are postulated to play a role in the pathogenesis of several vasculitis illnesses and infectious diseases. To investigate the presence of cryoprecipitates in Kawasaki syndrome, we studied sera from 25 children with acute Kawasaki syndrome. None of the subjects was treated with intravenous gamma-globulin. Cryoprecipitates were detectable in sera of 11 of 25 (44%) children studied. The mean (+/- SE protein concentration of the cryoprecipitates was 88.0 (+/- 20.2) micrograms/ml serum. Cryoprecipitates consisted primarily of IgG and IgM; no complement components were detected but highly sensitive methods were not used. The presence of cryoprecipitates in the serum of children with acute Kawasaki syndrome was associated with the subsequent development of coronary artery aneurysms detected by echocardiogram (P less than 0.05). There was no association between detectable cryoprecipitates and either peak platelet count or erythrocyte sedimentation rate. In one patient, measurement of cryoprecipitates in serial samples showed a reduction in concentrations that paralleled subsidence of disease activity. We speculate that cryoprecipitates may be a marker for increased risk of coronary aneurysm formation and may play a role in the pathogenesis of the cardiac disease in Kawasaki syndrome.

An in vitro assay for radiographic contrast media idiosyncrasy.

Adverse reactions related to the use of iodine-containing radiographic contrast media (RCM) continue to present a significant problem in diagnostic radiology. This study was designed to test an in vitro assay that can probably be used before infusion of RCM to predict patients with high risk of developing contrast media reactions. Peripheral leukocytes of 23 patients with a positive history of RCM incompatibility or idiosyncrasy, 89 atopic individuals, and 49 normal, nonatopic volunteers were stimulated in vitro with three RCM in different concentrations, and the amount of histamine released was measured in the supernatant. There was a significant increase in histamine release induced by RCM in the majority of patients with RCM idiosyncrasy, and in patients with atopic disease. Normal leukocytes, when incubated with serum from high-releasing or atopic patients, did not show increased histamine release after stimulation with the respective RCM. The increased releasability of patients' leukocytes could not be transferred by serum. Our results show that leukocytes' histamine release is higher in patients with a positive history of RCM incompatibility and patients with atopic disease. This in vitro assay might be a useful diagnostic tool for detecting patients with a high risk of developing contrast media reactions.

Screening test for complement activation by counterimmunoelectrophoresis.

This report describes the use of counterimmunoelectrophoresis (CIE) as a method of detecting activation of the complement system. With this technic small amounts of C3 split products (C3c/d) can be detected in plasma samples by specific precipitation with antiserum in less than two hours. The CIE technic is a highly sensitive, rapid method for detecting activation of the complement system in the presence of normal concentrations of C3 measured hemolytically or by radial immunodiffusion (RID) in human disease. A clinical investigation was carried out in 40 patients with systemic rheumatic diseases and 116 normal healthy individuals. The following observations were made: (1) plasmas and sera from normal individuals had normal total complement hemolytic activity (CH50), hemolytic active C4 (C4H50) and C3 (C3H50); (2) in 30% of the serum samples it was possible to identify the presence of C3 split products, in contrast to only 2.5% of the plasma samples obtained simultaneously; (3) in the specimens from patients who had rheumatic disease activity, C3 split products were identified by CIE in all cases except one in the presence of normal C3 protein measured by RID.

Herpes gestationis associated with the C3 nephritic factor.

Herpes gestationis (HG) is a rare, blistering disease of pregnancy and the puerperium that has immunologic findings of C3 with or without IgG deposited in a linear, band-like distribution along the dermoepidermal basement membrane zone (BMZ). A circulating IgG factor has been demonstrated that will fix complement by the classic pathway in vitro. This study involves a patient who had classic HG with C3 at the BMZ, and who had a circulating factor with activity via the alternative complement pathway similar to the previously described C3 nephritic factor found in patients with partial lipodystrophy, systemic lupus erythematosus, and membranoproliferative glomerulonephritis. Like the C3 nephritic factor, the activity was detected in IgG fractions of serum samples.

Plasma complement and histamine changes after intravenous administration of sodium fluorescein.

Venous blood samples were obtained from 85 patients before and at three, ten, and 30 minutes after intravenous administration of sodium fluorescein. Whole complement hemolytic activity and plasma histamine were measured. The patients were observed for side effects. Clinical adverse reactions occurred in 18 (21%) of the patients. Changes in complement hemolytic activity occurred in all patients, but probably were the result of binding of fluorescein to complement proteins, or on the membrane of sensitized red blood cells used in the assay. Plasma histamine was increased within the first few minutes after infusion of fluorescein and persisted up to ten minutes. Increased histamine was found in 66% of patients with adverse reactions and in only 15% of patients with no reactions. Histamine may be an important mediator of adverse reactions to fluorescein.

[Myocardiopathy diagnosed in utero in a mother with SS-A antibodies treated with plasmapheresis]. / Miocardiopatía diagnosticada in utero en madre con anticuerpos contra SS-A tratada con plasmaféresis.

We report a 36 years old patient with Sjogren's syndrome, who during her second pregnancy, the product developed a miocardiopathy with complete heart block that was diagnosed in utero at 26 weeks of pregnancy. Simultaneously, laboratory data reported a SS-A/Ro titer of 1:50,000 with positive antiphospholipids antibodies. Patient was subjected three times to plasmapheresis with three blood volume exchange each time. During the procedures, we had monitor the product and no hemodinamic changes were observed. Unfortunately, 25 days later the patient reported absence of fetal movement and by ecosonography and Doppler was not observed fetal movement or cardiac function. This pregnancy ends in cesarea. The patient is in perfect clinical conditions under control using prednisone and methotrexate.

[The rapid diagnosis of rubella and measles by the dot immunoassay method]. / Diagnóstico rápido de rubéola y sarampión por el método de inmunoensayo en mancha.

A procedure is described for the routine laboratory identification of IgM or/and IgG antibodies to measles and rubella simultaneously. A rapid dot-immunobinding assay on nitrocellulose was compared with ELISA systems. 58 serum samples from patients with an exanthematous disease and 15 serum negative samples for both virus were studied. The type of antibody found in all samples was variable and was always related to the presence of immunity in each individual. However, all known negative samples were negative. When results were compared with commercially available ELISA systems or reference laboratories, we found an excellent correlationship including titers. We concluded that the dot-immunobinding assay is a rapid, specific, sensitive, reproducible and economic test that can be used simultaneously for identification of several antigens.

[Recurrent otitis media with effusion and food allergy in pediatric patients]. / Otitis media recurrente con efusión y alergia a los alimentos en pacientes pediátricos.

INTRODUCTION: Recurrent otitis media with effusion continues to be important pediatric clinical problem and is related to food allergy. OBJECTIVE: The purpose of the present investigation was to determine if the recurrent otitis media with effusion was associated to food allergy. METHODS: We reviewed medical charts from children with food allergy and otitis media with effusion for a period of three months of duration or every month for the last six months seen in an allergy clinic. Patients with anatomic abnormalities, polypous or immunologic deficiencies were excluded. Every patient was challenged with foods to which he/she was allergic in order to demonstrate cause-effect. In all patients we performed tympanometries. RESULTS: We found twenty five patients with recurrent otitis media with effusion and food allergy demonstrated by positive skin testing. The most common food found to be associated was milk, egg, beans, citrus, and tomato. The elimination of the food diet led to a significant amelioration of the otitis in 22 patients, in whom clinical and tympanometry evaluation was performed. The challenge diet with suspected offending food provoked a recurrence of the otitis problem. CONCLUSION: These results demonstrated the association between recurrent otitis media with effusion and food allergy. Therefore, all patients with recurrent otitis media with effusion should be investigated for food allergy.

[Klebsiella pneumoniae glycoproteins as coadjuvants in the prevention of recurrent respiratory infections in children 1-6 years of age]. / Glicoproteínas de Klebsiella pneumoniae como coadyuvantes en la prevención de infecciones recurrentes respiratorias en niños de 1 a 6 años de edad.

MATERIAL AND METHOD: Forty two children with clinical diagnosis of recurrent high and low respiratory infections, were treated with an extract of P1 and F1 glycoproteins; fraction of Klebsiella pneumoniae, 1 g/day for one week a month for three months, according to a double-blind, randomized, parallel group trial. RESULTS: Improvement of clinical and laboratory results were compared with 44 control patients with the same age and similar sex distribution, that were treated with placebo. At the end of study, clinical analysis at 6, 9 and 12 months after the initial dose of Klebsiella pneumoniae, decreased in the number of respiratory infections, sick days, and school assistance were observed in the study group when compared with the control group (p < 0.001 in all parameters). There were not statistical significant changes in hemolytic complement, C3, C4 and immunoglobulins (IgA, IgG, IgM and IgE) levels before and after treatment and between groups. There were not observed adverse reactions to oral treatment with this extract of Klebsiella pneumoniae. CONCLUSIONS: These results suggested that the extract of Klebsiella can be used as a coadjuvant in the treatment of pediatric patients with recurrent high and low respiratory infections.

The complement system in pediatric renal disease.

A brief review of the classical and the alternative pathways of complement activation is presented. Clinically, according to the complement system, we can divide the children with glomerulonephritis into two groups, normocomplementemic and hypocomplementemic. In addition, inherited complement deficiencies can be identified associated with renal diseases. We discuss the three possible sources of complement in urine, although more control studies are necessary in patients with different causes of proteinuria in order to define the clinical significance of complementuria. The immunohistological results of glomerular nephritic biopsy material by the fluorescence antibody technique is analyzed with respect to clinical diagnosis and evaluation of the treatment. The nature of C3NeF as an antibody to factor B-C3 complex is demonstrated by different groups and in different diseases. Finally, the presence of a receptor for complement in the glomerulus, is explained in human disease by the deposition of immune complexes into the renal glomeruli.

The complement system of the newborn infant.

Whole complement activity (CH50), and levels of some components of the classical (C1q, C4, C3) and alternative (factor B and properdin) pathways were determined in 55 non-infected and 11 infected newborn infants. Normal newborn infants were lower than adults in all complement measurements; preterm infants being significantly lower than term infants. Complement was not effected by intrauterine growth retardation. Absence of C3 split products indicated that the deficiencies were developmental and not due to activation of the complement system. Complement levels were lower in infected infants and this was due to activation of the complement system as C3 split products were present in 54%. Because of the high incidence of split products in infected infants, incorporating a test to determine their presence may be of benefit in the diagnosis of the presence of infection in newborn infants.

Alteration of human blood cells and changes in plasma mediators produced by radiographic contrast media.

In vitro incubation of human blood cells with iodinated radiographic contrast media (RCM) produced marked effects which were dose-dependent: erythrocytes showed crenation which was reversible; neutrophil leukocytes released the lysosomal enzyme beta-glucuronidase; basophil leukocytes released histamine; and platelets released serotonin as well as beta-glucuronidase. The release reaction could not be attributed to cell lysis, as demonstrated by the release of the cytoplasmic enzyme lactic dehydrogenase (LDH). In normal human serum, RCM produced activation of the complement system with lysis of cells. This RCM-induced complement activation seemed to occur via the alternate pathway. Stabilizers and cations present in the clinically used RCM solutions did not produce any complement changes.

Mechanism of complement activation by radiographic contrast media.

Activation of the complement system by radiographic contrast media (RCM) was demonstrated by in vitro haemolytic and immunological assays. Such activation was found to be a function of the RCM molar concentration. Iodipamide was the most active of five RCM tested. When RCM was incubated with normal human serum (NHS) in the presence of ethylene glycol-tetra-acetic acid and magnesium ions, conditions which block activation of the classical pathway but permit activation of the alternative pathway, haemolytically active C3, properdin and factor B were found to be decreased but haemolytically active C4 was normal. Using counterimmunoelectrophoresis, the activation of complement was further demonstrated by detection of C3 and factor B-split products. Finally, when radiolabelled complement proteins were reacted with RCM in vitro and studied by density-gradient ultracentrifugation, it was demonstrated that a large complex was formed with a sedimentation of 22S, similar in characteristics to the C5b-C9 complex. It was postulated that the mechanisms of in vitro consumption of complement by RCM was mainly through the alternative pathway.

[Identification of antinuclear, nucleolar and SS-A antibodies, using an immunoassay system]. / Identificación de anticuerpos antinucleares, nucleolares y SS-A, con el uso de un sistema de inmunoensayo.

In this paper we report a solid phase ELISA for screening of antinuclear, nucleolar, and SS-A antibodies. The test system was evaluated with 158 sera that were positive by immunofluorescence (IF), some of which were immunologically characterized as containing antibodies to Sm, RNP, SS-A, SS-B, nucleolar, and DNA, and 247 IF negative that were used as a control group. Ninety eight per cent of serum samples with an antibody of known specificity and/or positive IF, were positive by the ELISA system including antinucleolar samples. Reproducibility of the assay was demonstrated by using five sequentially prepared antigenic extracts. The lineal regression from the last experiment was between 0.75 and 0.91. With this system, positive and negative ANA can be identified as the screening procedure without the need of additional studies. To our knowledge this is the first ELISA system report for the screening of antinucleolar antibodies.

Activation of the alternative pathway of complement by antiserum to factor B.

Monospecific rabbit and goat antisera to human complement proteins and human immunoglobulins were tested for their ability to activate the alternative complement pathway. This activation was detected by two methods where classical pathway activation was blocked with EGTA and alternative pathway activation was promoted with added magnesium ions. These two methods consisted of lysis of GSHE and conversion of factor B into split products. C1q-depleted serum was used in a third assay system. Only antiserum to human factor B was able to activate the alternative pathway in the various systems used. None of the other anticomplement sera showed such activity. When antiserum to factor B was fractionated by ammonium sulfate and column chromatography, activation of the alternative pathway was found in the IgG fraction, and this activity was completely removed by absorption with purified factor B but not with other purified complement components.

Genetic deficiency of the fourth component of complement (C4) in Wistar rats.

A chance observation has led to the discovery of single component complement deficiency in Wistar rats. During serial total haemolytic activity (CH50) determinations on 10 male rats, 2 were consistently found to have values 20% or less than that of the others. An additional 30 males (200--220 g) and 20 females (180--200 g) were screened for CH50 and the defect was found in 10 of the males but none of the females. Mixtures of sera of high and low haemolytic activity gave intermediate values between the two populations arguing against the presence of an inhibitor of the complement system. Single component analysis revealed the defect to reside in the fourth component of complement (C4). Highly purified human C4 completely reconstituted the haemolytic activity of the complement deficient rat serum. A linear relationship between C4 and CH50 titres was observed. Data from preliminary selective breeding experiments point toward an autosomal recessive mode of inheritance. Animals with intermediate values were observed indicating a gene dosage effect with which the heterozygote may be recognized.