Demonstration and quantitation of activation of the first component of complement in human serum.

Activation of the first component of human complement (C1) in human sera can be readily detected in double immunodiffusion studies with anti-C1q, anti- C1r, and anti-C1s as it produces a characteristic pattern quite different from that of precursor C1. Native macromolecular C1 gives a continuous line of precipitation with antisera to C1q, C1r, and C1s in double diffusion studies. After activation of C1 by incubation of serum with complement activators, three major changes occurred in the Ouchterlony pattern. First, spurring of the C1s precipitin line over that of macromolecular C1, indicating release of C1s from C1, was observed with low doses of activator. Release of C1s was quantitated by single radial diffusion and shown to be complete with the highest activator dose examined. Second, C1q was released with larger activator doses as shown also by spurring of the precipitin line due to this component over the remaining macromolecular C1. Third, and most surprising, C1r antigenicity was progressively lost as the activator dose was increased and no C1r line remained with the highest dose of activator tested. This was not true with C1s as there was no change in the total C1s concentration in serum incubated with various activator doses. These observations provide two approaches to the quantitation of C1 activation in human serum. First, C1r and C1s can be quantitated by single radial diffusion. A decrease in the C1r:C1s ratio correlates with activation. Second, C1s released by the activation can be quantitated by single radial diffusion if the agarose contains high concentrations of anti-C1q to confine C1, also containing C1s, to the area near the application well, and lesser concentrations of anti-C1s to permit free C1s to produce a measurable ring. The extent of release of C1s also correlates with activation. These immunochemical techniques to quantitate C1 activation directly inserum do not require specialized reagents. It is hoped that they will be useful in screening pathological sera and in monitoring the status of the complement system in patients.

Direct demonstration and quantitation of the first complement component in human serum.

The first component of complement, C1, can be demonstrated and quantitated in normal and pathological human serums by simple immunochemical techniques. All of the C1q, C1r, and C1s detected in normal serum was found to be in the C1 complex. A simple modification of these methods permitted the quantitation of free C1s in the presence of macromolecular C1, a technique which may prove useful in screening pathological serums.

Hereditary angioedema in a family presenting as transient periarthritis.

Hereditary angioedema (HAE) is a rare condition known to cause episodic, self-limiting, nonpruritic, nonpitting edema that involves skin and visceral organs. It may affect any external body surface including face, extremities, and genitalia. Most commonly involved viscera are gastrointestinal and respiratory systems. Patients may have severe abdominal pain because of edema of the bowel wall. This disease can cause life threatening laryngeal edema if it involves the airway.We describe a patient with HAE who was initially diagnosed with arthritis after she had recurrent edema around her peripheral joints. Diagnosis of HAE in her led to the same diagnosis in her sister and her father. HAE should be considered in the differential diagnosis of recurrent attacks of periarticular swelling.

Circulating immune complexes in sera of children with neuroblastoma: correlation with stage of disease.

The presence of circulating immune complexes (ICS) in freshly drawn sera of 67 children with neuroblastoma was studied by the Raji cell radioimmunoassay of Theofilopoulos et al. (J. Clin. Invest. 57: 169--182), with particular emphasis on the correlation of levels of ICS with stage of disease and changes attributable to treatment. There was a close correlation between amount of complexes and stage of disease and treatment. Levels of ICS increased as the stage of the disease advanced, and were significantly higher (P less than 0.005) in stage IV than in all other stages combined. When patients with stage IV disease were subdivided into "before," "during," and "after" treatment groups, there was a significant decrease in ICS levels as treatment progressed. Studies of complement and complement components did not give such a clear relationship. A significant decrease of hemolytic C1 values was found in patients with "active disease" compared to normal age-matched controls. Some high C3 levels, determined immunochemically, were associated with low hemolytic levels of C3, which were attributed to C3 cleavage detected by immunoelectrophoresis. Based on our survival data, ICS, which were significantly different in 20 patients now decreased when compared to those of other patients, are very valuable in the prognosis of neuroblastoma.

Immunoglobulins and complement components in synovial fluid of patients with acute rheumatic fever.

Three components of complement and six other serum proteins were assayed in synovial fluid and serum samples from 25 patients with acute rheumatic fever in Trinidad. The resulting data indicate a relative decrease in both early and late components of complement within the synovial fluids which suggests local activation by immune complexes. Such activation of complement within the joint spaces may play a primary role in development of the inflammatory arthritis of acute rheumatic fever.

[The range and potential contribution of irreversibly adsorbed proteins on the surface of artificial implants]. / Spektr i potentsial'naia rol' neobratimo adsorbirovannykh belkov na poverkhnosti iskusstvennykh implantiruemykh materialov.

Protein adsorption is the first stage of the interaction between prosthetic materials with tissues of the body. They undergo conformational changes depending on the chemical composition and the nanotopography surface. Adsorbed proteins induce adhesion and alter the functional state of migrating cells. Plasma samples from patients were incubated with such matrices as titanium, polypropylene or polyester with fluoropolymer coating meshes. Bound peptides were analyzed by electrophoresis. Qualitative analysis of the peptides extracted from the gel was performed by chromatography-mass spectrometry. Quantitative analysis was performed by the MRM method. More than 60 proteins were identified on the analyzed surfaces. Quantitative analysis showed preferential adsorption of vitronectin, albumin, fibrinogen a-chain, C1ѕ component of the complement system. Vitronectin had the maximum relative protein content. Since biocompatibility of the analyzed materials varies considerably this variability may be attributed to conformational changes occurring with vitronectin during its irreversible adsorption.

Predictive value of serum anti-C1q antibody levels in experimental autoimmune myasthenia gravis.

Components of the complement cascade and circulating immune complexes play important roles in both experimental autoimmune myasthenia gravis and myasthenia gravis in humans. Thus far, no serological factor has been identified to predict the clinical severity of either myasthenia gravis. Upon immunization with acetylcholine receptor, levels of complement factors C1q, C3 and CIC increase with time in sera from C57BL/6 (B6) mice. Both these and plasma samples from myasthenia gravis patients also contain anti-C1q antibodies. The serum levels of anti-C1q antibodies but not C1q, C3 and CIC are significantly correlated with the clinical severity in the experimental myasthenia mice. However, this correlation is not observed in myasthenia gravis patients.

Association of levels of circulating C1q binding macromolecules with induction chemotherapy response in head and neck cancer patients.

Ninety-five untreated patients with squamous cell carcinoma of the upper aerodigestive tract expressed significantly higher levels of C1q-binding macromolecules as compared to 45 noncancer-bearing controls. No relationship between C1q-binding macromolecules and levels of circulating IgG-immune complexes as determined by the solid-phase C1q-binding assay or the C3d-solid-phase assay could be defined suggesting that C1q-binding macromolecules were distinct from IgG-circulating immune complexes. An elevated level of C1q-binding macromolecules within these patients was predictive of subsequent response to induction chemotherapy; those with elevated levels characteristically showed no response. Using multivariate logistic regression analysis including the covariates of American Joint Committee staging parameters as well as C1q assay results, levels of the isolated macromolecules added significant prognostic information as to the probability of chemotherapeutic response. The quantitation of C1q macromolecules has clinical implication as to choice of therapeutic regimens against head and neck cancer. The nature of these substances remains to be defined.

Studies of urticaria and acute serum sickness with the C1q precipitin test.

The C1q precipitin test was performed in serum samples from five groups of patients: (1) 20 patients with acomplementemic systemic lupus erythematosus glomerulonephritis (SLE), (2) 2 patients with serum sickness due to the administration of horse serum, (3) 2 patients with serum sickness preceding hepatitis B, (4) 50 patients with chronic urticaria, and (5) 30 normal controls. Positive C1q precipitin tests were found in all patients with SLE and the four cases of serum sickness. Positive tests correlated with depressed serum complement (C3 and C4) levels and were found only in the early phase of serum sickness. Urticaria patients uniformly had negative C1q precipitin tests and normal serum complement levels.

Immune complexes, complement, and anti-DNA in exacerbations of systemic lupus erythematosus (SLE).

The usefulness of serological parameters in assessing clinical exacerbations of SLE was examined. Patients with active renal disease tend to have lower levels of CH50 and C3 and highest levels of immune complexes detected by C1qBA than those patients with extrarenal manifestations only. Patients with a combination of both active extrarenal and renal disease are more likely to demonstrate the lowest levels of CH50, C4, and C3. However, immune complex levels are not higher than levels detected in patients with only active nephritis. A normal C3 level argues against active nephritis. Low complement levels without appreciably elevated levels of C1qBA suggest that significant renal disease is unlikely. The serial measurements that best reflect evolving clinical activity and which may serve as markers of impending exacerbation are, in decreasing order of usefulness: C4, CH50, C1qBA, C4, C3 and ADA. However, a combination of CH50, C4, C3 and C1qBA appeared to be the most useful. Given various serologic changes, guidelines for following patients are offered.

Relationship between renal pathology and the size of circulating immune complexes in patients with systemic lupus erythematosus.

Sera from 35 patients with biopsy-proven diffuse proliferative (WHO class IV) or membranous (WHO class V) lupus nephritis were analyzed for the presence and size of circulating immune complexes. Elevations of the C1q solid-phase assay (C1qSP) for immune complexes were found in sera from all patients with diffuse proliferative nephritis, with a mean +/- 1 SEM of 166.8 +/- 42.0 micrograms/AHG-equivalents/ml serum, and in 71.4% of the patients with membranous nephritis (83.1 +/- 26.7, p = 0.06). Using the WHO criteria for subclasses of membranous lupus nephritis, we also designated renal biopsies as nonproliferative (WHO classes Va and Vb) or proliferative (WHO classes IV and Vc). Employing the latter groupings, we observed significant differences between C1qSP results of patients with nonproliferative (30.3 +/- 8.8) and proliferative (172.8 +/- 36.8, p less than 0.001) lupus nephritis. These data suggest that the presence of C1q-binding material in serum is pathophysiologically related to proliferative glomerular lesions, and that levels of C1qSP binding reflect renal lesions in SLE patients. Sucrose density gradient ultracentrifugation was performed on each serum, and gradient fractions analyzed for C1qSP-binding and total IgG, using techniques to minimize losses of immune complexes. The predominant peak of C1qSP activity sedimented with the 6.6S monomeric IgG. The 6.6S C1q-binding IgG was increased only in 1 of 10 patients with membranous lupus nephritis without proliferative changes, and was elevated in 16 of 25 patients with proliferative lesions (WHO classes IV and Vc). A significant negative correlation was found between the presence of this C1q-binding material and subepithelial electron-dense deposits, suggesting that the presence of this material contributed to the absence of subepithelial immune deposits. Large-molecular-weight C1qSP-binding material was also present, mainly in sera from patients with proliferative lesions. Furthermore, highly positive correlations were found between immune deposits in interstitial blood vessels and peritubular areas, and the concentrations of C1qSP-binding IgG and rapidly sedimenting IgG in density gradient analysis. Overall, these findings are consistent with the hypotheses that circulating immune complexes contribute to the pathogenesis of glomerulonephritis and interstitial nephritis in patients with SLE, and that 6.6S C1q-binding IgG plays a role in the proliferative lesions of lupus glomerulonephritis.

Studies on the occurrence of circulating immune complexes in vascular diseases.

The presence of circulating immune complexes was studied in 347 samples of serum from 212 patients with various vascular diseases. Two quantitative methods (complement-consumption assay and C1q-solubility test) were used for the measurement of the concentration of the complexes. Immune complexes were detected in each group of patients tested (coronary arteriosclerosis, myocardial infarction, cerebral artery sclerosis, arteriosclerosis obliterans, phlebothrombosis, pulmonary infarction). A high proportion of positivity was recorded in myocardial infarction (in 43 patients out of the 94 tested) and in arteriosclerosis obliterans (7 out of 11 cases). The possible pathogenic role of the circulating immune complexes is discussed.

Detection of C1q-bearing immune complexes by a monoclonal anti-C1q ELISA system.

A monoclonal antibody directed against the collagenous portion of human C1q was used to detect C1q-bearing immune complexes in patients with rheumatic disorders. Sera of patients with rheumatoid arthritis, systemic lupus erythematosus (SLE), osteoarthritis, as well as normal human sera (NHS) used as controls were tested in an ELISA system. C1q-bearing immune complexes were bound to a solid-phase monoclonal anti-C1q antibody, and detected with F(ab')2 antibodies to human IgG. Heat-aggregated human IgG was adjusted to the same concentration as the WHO standard for immune complexes and used for the standard curve in NHS. The mean value in NHS was 19.5 micrograms/ml equivalents of aggregated IgG. Using 2 SD over the mean as the upper limit for normal values, samples greater than 43 micrograms/ml were considered positive. Patients with osteoarthritis were negative; high levels of C1q-bearing immune complexes were detected in patients with rheumatoid arthritis (up to 800 micrograms/ml equivalents of aggregated IgG). With our assay C1q-bearing immune complexes were detected with high frequency (81%) in the sera of patients with rheumatoid arthritis, while a C1q solid-phase binding assay (C1q SPBA) revealed positive results only in 67% of rheumatoid arthritis sera. Compared to NHS, CH50 titers and C1q values of sera from patients with rheumatoid arthritis were frequently high. In contrast, the sera of SLE patients with low CH50 titers and low C1q levels had IgG immune complexes which could be detected only in the C1q-SPBA. C1q-bearing immune complexes were not detectable in the sera of patients with SLE. Since C1q triggers activation of the classical C pathway, this assay with monoclonal anti-C1q antibody appears to be useful for detecting immune complexes in rheumatoid arthritis patients with normal or elevated CH50 and C1q values, especially in the early stage of the disease.

An ELISA technique for the measurement of C1q in cerebrospinal fluid.

Determination of the C1q content of cerebrospinal fluid (CSF) may be of value in understanding the immunological reactions occurring within the central nervous system (CNS). A double sandwich ELISA method has been developed for the detection of C1q in human serum and CSF. It uses polyclonal antibodies and is sensitive in the nanogram range. The mean concentrations of C1q were determined to be 127 micrograms/ml in serum and 0.4 microgram/ml in CSF. These results suggest that increased levels of C1q in the CSF play a role in some neurodegenerative disorders.

A substrate amplification system for enzyme-linked immunoassays. Demonstration of its general applicability to ELISA systems for detecting antibodies and immune complexes.

Self recently described a substrate system for alkaline phosphatase (AP)-dependent ELISAs which markedly increased sensitivity, compared to using p-nitrophenyl phosphate. This increase is achieved by having AP, the primary enzyme, produce an activator for a secondary enzyme-substrate system, within which marked amplification occurs. We adapted this technique to study antibodies to casein, bovine serum albumin, ovalbumin, and cardiolipin in the sera of patients with systemic lupus erythematosus (SLE) and normal individuals. The new substrate system yielded titres 30-50-fold higher than those with p-nitrophenyl phosphate (Sigma 104, p-NPP). In addition, when used in a solid-phase C1q binding assay we were able to use a 1 : 100,000 dilution of AP-conjugated anti-human IgG with the amplified substrate, compared to the 1 : 1000 dilution needed with p-NPP. This system is extremely valuable because of its flexibility. It can either be very sparing of limited samples, or if the added sensitivity is not needed, 100-fold less AP conjugate may be used. Thus rare or expensive conjugates can be significantly conserved.

Inherited deficiency of the second component of complement (C2) with membranoproliferative glomerulonephritis.

The occurrence of membranoproliferative glomerulonephritis in a 13 year old boy with inherited complete deficiency of the second component of complement (C2) is described here for the first time. Results of the complement studies and the associations of glomerulonephritis with complement deficiencies are discussed.

Immunologically-mediated acute renal failure of nonglomerular origin in the course of systemic lupus erythematosus [SLE]. Report of two cases.

Acute anuric renal failure was observed in two patients with systemic lupus erythematosus (SLE) during the clinical and serologic active phase of the disease. Renal biopsies, performed during the acute episodes, showed only mild and focal mesangial cell proliferation without deposits. In contrast, tubulointerstitial lesions were predominant. Intense granular immune deposits along the tubular basement membrane, or immunofluorescence examination, were suggestive of immune complex deposition. One of these patients had severe high blood pressure and vascular lesions likely induced by immune complexes. In both, renal function was recovered. Immunologically-mediated tubular and vascular lesions in the course of SLE are discussed.

Circulating IgA-immune complexes in Henoch-Schönlein purpura. A longitudinal study of their relationship to disease activity and vascular deposition of IgA.

In Henoch-Schönlein purpura immune complexes in inflamed vessel walls characteristically contain immunoglobulin A(IgA). To determine whether IgA is also the predominant immunoglobulin in circulating immune complexes, we compared the results of three immune complex assays with specificities for different classes of immunoglobulins in a longitudinal study of 37 patients (30 children and seven adults) with Henoch-Schönlein purpura. Circulating IgA-containing immune complexes were detected by their reactivity with a low avidity anti-IgA antibody in 27 of the 37 patients. IgA was simultaneously present in cutaneous vessel walls in 95 percent of the patients with circulating IgA-containing immune complexes. High levels of IgA-containing immune complexes were found only during the initial phase of the disease. Immune complexes containing bound complement breakdown products were demonstrated by binding to conglutinin. IgA was found in these immune complexes in 17 patients, IgG in 17 and IgM in nine patients. There was no apparent relation with the class of immunoglobulin in the deposits. Conglutinin-binding immune complexes were present later in the course of the disease and after remission. C1q-binding immune complexes were only found in two patients. These findings suggest that immune complexes-containing IgA may initiate the vasculitis of Henoch-Schönlein purpura, whereas complement-reacted immune complexes containing immunoglobulins of the other classes appear in the circulation in a later phase.

Elevated plasma levels of the anaphylatoxins C3a and C4a are associated with a fatal outcome in sepsis.

PURPOSE AND PATIENTS AND METHODS: Both complement and contact system of coagulation have been implicated in the pathophysiology of sepsis. We therefore measured levels of the complement activation products C1-C1-inhibitor complexes and C3a in serial plasma samples (obtained every six hours) from 48 patients with clinically suspected sepsis, and related these levels to the clinical outcome. C4a was also measured in samples obtained on admission. RESULTS: C3a levels were elevated in 47 patients at least once during the observation period. These levels appeared to be considerably higher in patients who died than in patients who survived. This difference was found for the levels on admission (p = 0.0003), as well as for the highest (p = 0.0010) and the lowest (p less than 0.0001) levels encountered in each patient. The mortality in patients with plasma C3a levels of 13 nmol/liter or less on admission (27 patients) was 33 percent, compared with 86 percent in patients with levels of 14 nmol/liter or more. Patients with septic shock had significantly higher C3a levels than normotensive patients (p values between 0.046 and 0.004). No significant differences in C3a were found between patients who had respiratory distress syndrome and those who did not. C4a levels in plasma samples obtained on admission were elevated in 43 patients. These levels correlated very significantly with C3a levels (p less than 0.0001), and showed similar associations with a fatal outcome. C1-C1-inhibitor complexes were elevated in 23 patients at least once during the observation period. These patients had significantly higher levels of C4a and C3a than patients with normal amounts of C1-C1-inhibitor complexes. Patients who died had higher levels of C1-C1-inhibitor complexes than patients who survived. However, this difference was not significant. CONCLUSION: On the basis of our results, we propose that activation of the complement system via the classical pathway is involved in the development of fatal complications in sepsis.

Complement activation and CD59 expression in the motor facial nucleus following intracranial transection of the facial nerve in the adult rat.

Intracranial transection of the facial nerve has been shown to cause a massive neuronal cell death in the motor facial nucleus. Complement activation has been proposed to contribute to neuronal degeneration following axotomy. Using immunocytochemistry and in situ hybridization we show in the present study that there is complement activation in the facial nucleus after intracranial facial nerve transection as well as increase of the complement regulators CD59 and clusterin. We propose a neuroprotective role for the complement regulators CD59 and clusterin against homologous attack of complement to facial motor neurons.