The role of complement activation in the pathogenesis of Fuchs' dystrophy.

PURPOSE: Inflammation can be an etiologic factor of Fuchs' dystrophy according to previous studies. Our aim was to analyse the activation of the complement system in the aqueous humor in this pathological condition. METHODS: 100 µl aqueous humor sample was taken during keratoplasty of 11 Fuchs' dystrophic patients and during phacoemulsification surgery of 18 control patients. The samples were mixed with EDTA and stored at -80 °C. Concentrations of C1rC1sC1Inh and C3bBbP complexes as markers of the activation of the classical and alternative complement pathways, respectively, were measured with ELISA method. The results of the patient group and the control group were compared with statistical analysis (non-parametric Mann Whitney test). RESULTS: Both the concentrations of C1rC1sC1Inh [4.3 (3.2-20.2)AU/ml] and of C3bBbP [15.3 (7.8-22.6)AU/ml] were significantly higher in the Fuchs' dystrophic group than in the control group [C1rC1sC1Inh: 0.0 (0.0-5.6)AU/ml, C3bBbP: 1.4 (0.0-7.8)AU/ml]. The median value is shown along with the (25% and 75% percentiles). CONCLUSIONS: Based on our results, the complement system may be activated both through the classical and alternative pathways in the aqueous humor of the patients with Fuchs' dystrophy.

Elevated C1rC1sC1inh levels independently predict atherosclerotic coronary heart disease.

UNLABELLED: Clinical studies as well as animal models emphasized the importance of the complement system in the pathogenesis of coronary atherosclerosis and cardiovascular diseases. Our aim was to examine the extent and clinical implication of complement system activation in patients with stable atherosclerotic coronary heart disease (ACHD). Seventy-six patients with stable angina pectoris (SAP) scheduled for elective coronary angiography were enrolled into the study. Percutaneous coronary intervention (PCI) was performed in 24 patients, in 27 patients (NOPCI group) the coronary angiography showed significant stenosis and bypass surgery (CABG) or optimal medical therapy (OMT) were advised, whereas in 25 patients the coronary angiography was negative (NC group). 115 volunteers served as healthy controls (HC). In all individuals, the plasma level of several complement activation products - C1rC1sC1inh, C3bBbP and SC5b-9 - were determined on admission, strictly before the coronary angiography. In patients with angiographically proven ACHD (PCI and NOPCI groups), the baseline C1rC1sC1inh levels were significantly higher compared to NC group and HC (p<0.0001, for both comparisons). According to the multiple logistic regression analysis, high C1rC1sC1inh level proved to be an independent biomarker of coronary heart disease (p<0.026, OR: 65.3, CI: 1.628-2616.284). CONCLUSION: Activation of the classical complement pathway can be observed in angiographically proven coronary atherosclerosis. Elevated C1rC1sC1inh levels might represent an useful biomarker for coronary artery disease.

Proteomic analysis of plasma from Holstein cows testing positive for Mycobacterium avium subsp. paratuberculosis (MAP).

Johne's disease (JD) is a widespread and economically important chronic inflammatory disease of the small intestine of ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP). Although there are several techniques available for diagnosis of JD, their sensitivity is questionable. New proteome profiling methods, such as serum/plasma protein fingerprinting by 2-Dimensional Fluorescence Difference Gel Electrophoresis (2D-DIGE), may therefore be useful for identifying novel protein biomarkers of MAP infection. In this study, plasma samples were collected from 380 Holstein cows and screened for the presence of MAP infection using the M.pt. Johne's antibody Kit (IDEXX). Five negative (MAP-), and 5 strongly positive (MAP+) cows were selected for proteomic analysis. Highly abundant proteins were depleted from the plasma samples using the ProteoMiner technology (Bio-Rad) to enhance the resolution of low abundance proteins. Plasma samples from MAP-, MAP+, and a pooled internal control were labelled with different fluorescent dyes and separated based on their isoelectrical point (IP) and then their molecular weight. Gel images of the fluorescent plasma protein maps were acquired using a Typhoon scanner and analyzed using the DeCyder software. Proteins that were differentially expressed were excised from the gels, trypsin digested, and subjected to MS/MS analysis for identification. Six proteins were identified as being up-regulated at least 2-fold in MAP+ cows including: transferrin, gelsolin isoforms α & ß (actin binding protein - ABP), complement subcomponent C1r, complement component C3, amine oxidase - copper containing 3 (AOC3), and coagulation factor II (thrombin) (p<0.05). Two proteins that were down-regulated approximately 2-fold in the MAP+ cows included coagulation factor XIII -B polypeptide (COAFXIII), and fibrinogen γ chain (FGG) and its precursor.

[Influence of serum IgG antibodies to Chlamydia trachomatis on the functional activity of complement components].

The immunoenzyme analysis and the method for the determination of IgG-containing immune complexes, carrying C1q component of the complement, were developed. In human blood sera the functional activity of components C3, complex C1r2s2, the content of C1 inhibitor and complement-activating immune complexes were determined. The comparative analysis of the activity of components C3 and C1r2s2, as well as between the content of C1 inhibitor and the activity of complex C1r2s2 for seropositive and seronegative sera, was made. Pronounced correlation for seropositive sera was observed. In addition, for seropositive sera correlation between an increase in IgG immune complexes and a drop in the functional activity of complex C1r2s2, as well as a drop in the functional activity of complex C1r2s2 and a growth in the titers of IgG antibodies to Chlamydia trachomatis, were established. The decreased functional activity of key complement components, simultaneously with the presence of complement-activating immune complexes and high titers of specific antibodies could be the diagnostic criteria of carrier state.

Usefulness of detection of complement activation products in evaluating SLE activity.

Complement activation products, such as C1rs-C1inh, specific for the activation of the classical pathway, C3b(Bb)P, specific for the activation of the alternative pathway and SC5b-9, specific for common terminal pathway of the complement cascade, were measured in healthy donors and in patients with clinically active and inactive systemic lupus erythematosus (SLE). Plasma levels of C3b(Bb)P and SC5b-9 were moderately, those of C1rs-C1inhibitor (C1rs-C1inh) were markedly elevated in patients with clinically inactive SLE, compared with healthy controls. The difference between active and inactive stages of the disease was best reflected by C3b(Bb)P plasma concentration (P<0.001), which also showed the highest correlation with the SLEDAI (Rs=0.41 P<0.001) and which was the most useful in distinguishing active and inactive sample pairs as well. The difference between SC5b-9 levels in the active and inactive stages was also significant (P=0.007), while that of C1rs-C1inh did not differ significantly (P=0.136). The correlation of the SLEDAI with SC5b-9 was 0.3 (P=0.015), while with C1rs-C1inh it was 0.21 (P=0. 089). These findings suggest that the measurement of complement activation products, especially that of the alternative pathway, are sensitive markers of the activity of SLE and can be used for clinical purposes.

SC5b-9 is the most sensitive marker in assessing disease activity in Brazilian SLE patients.

This study investigated whether increased plasma levels of terminal complement complex (SC5b-9) or split products correlate with disease activity and clinical manifestations in Brazilian systemic lupus erythematosus (SLE) patients. Comparisons with conventional measurements of complement and other inflammatory markers were also performed. Plasma levels of SC5b-9, C3a desArg, C1rs-C1Inhibitor, C3b(Bb)P, C3, C4, erythrocyte sedimentation rate (ESR) and mucoproteins (MP) were measured in 41 patients with SLE of different disease activity: 10 patients with none, 15 patients with mild, and 16 patients with moderate or severe activity. All parameters, with the exception of C3 and C3b(Bb)P, showed a statistically significant correlation with disease activity. Plasma levels of SC5b-9, C3a desArg, C4, CH50, ESR and MP revealed significant differences between the groups of patients without activity and those with moderate or severe disease. Although none of the variables were able to discriminate between patients without and those with mild activity, SC5b-9, C3a desArg, C4, ESR and mucoproteins showed significant differences between the patients with mild and those with moderate or severe disease. Among all the variables, SC5b-9 levels showed the most significant results and correlated well with the severity of the disease (p < 0.0005). Our data suggest that elevated levels of complement activation products, particularly of SC5b-9 are more sensitive markers in assessing disease activity than conventional laboratory diagnosis. Modern complement diagnosis is therefore recommended for monitoring disease progress in SLE patients.

Aspects of leukocyte function and the complement system following aerobic exercise in young female gymnasts.

Recent studies have reported reduced immunity in trained athletes. Scant information exists on changes in the immune function among trained children. The purpose of this study was to assess the effect of aerobic exercise on the phagocytic process of neutrophils and the complement system in young athletes. Subjects included prepubertal elite female gymnasts (n = 7) and untrained girls (n = 6) aged 10-12 years. Venous blood was withdrawn before, immediately post and 24 h following a 20-min run at a heart rate of 170-180 beats.min-1. Neutrophil random migration, chemotactic activity, bactericidal function and PMA/FMLP-stimulated superoxide anion release as well as various complement components were assessed. Net chemotaxis was found reduced (P < 0.05) 24 h following exercise (58 +/- 11 vs. 36 +/- 11 cells/field in gymnasts and 47 +/- 7 vs. 42 +/- 8 cells/field in untrained girls pre- and 24 h post-exercise, respectively). The basal values, as well as post-exercise values of bactericidal activity were lower (P < 0.05) in gymnasts as compared with the control group (0.8 +/- 0.3, 0.8 +/- 0.2 and 0.8 +/- 0.1 log decrease of colonies in gymnasts at pre-, immediately post-, and 24 h post-exercise, respectively and 1.1 +/- 0.1, 1.1 +/- 0.1 and 1.0 +/- 0.2 log decrease of colonies in controls, respectively). No significant effect on the bactericidal activity was observed in either group following exercise. The addition of homologous sera did not correct the bactericidal activity. PMA-stimulated superoxide anion release decreased (P < 0.05) among gymnasts immediately following exercise (5.7 +/- 0.4 vs. 4.4 +/- 1.0 mmol O2/10(6) PMN.min) and remained low 24 h later. The same trend was observed in FMLP-stimulated neutrophils but the data were not significant. Significantly decreased levels (P < 0.05) of the early complement components (C1Q, C1R) were also found following exercise (1.34 +/- 0.64 vs. 1.27 +/- 0.28 and 1.09 +/- 0.07 vs. 1.02 +/- 0.06 pre- and post-exercise in gymnasts and untrained, respectively). Furthermore, consistently lower C2 and C3 were observed in gymnasts compared with controls. Neutrophil dysfunction as well as impairment of the complement system seem to occur following exercise.

The human complement C1 complex has a picomolar dissociation constant at room temperature.

Periodic sampling of serum or reconstituted C1 initially diluted 1/2000 and 1/4000 (that is, to 0.1 and 0.05 nM) into a recombinant C1s-containing solution showed a gradual decline of hemolytic activity until equilibrium was approached, consistent with a simple dissociation, reassociation equilibrium, presumably C1 <--> C1q + C1r2C1s2. The presence of excess (5 nM) recombinant C1s minimized further dissociation of the C1r2C1s2, allowing the first step to be studied independently of the dissociation of C1r2C1s2 <--> C1r2 + 2 C1s. Reassociation experiments were also performed, starting with the dissociated C1 diluted to the same concentrations and following the regain of hemolytic activity to approximately the same values, showing that the same equilibrium had been achieved from both directions. Analysis of the kinetic data yielded forward and reverse rate constants and the equilibrium constant, for which values of approximately 72 and 3 pM were estimated at 0 and 23 degrees C, respectively. The effects of temperature, ionic strength, Ca2+ ion concentration, and activation of the zymogen on the equilibrium constants were explored; extreme sensitivity to temperature, ionic strength, and activation were found. At 23 and 30 degrees C, slow activation of C1 was also evident. Highly purified, reconstituted C1 yielded approximately the same values for the kinetic and equilibrium parameters as serum C1, suggesting that the structure of the reconstituted complex was similar to or identical with that of the serum C1 complex.

A neoepitope-based enzyme immunoassay for quantification of C1-inhibitor in complex with C1r and C1s.

Monoclonal antibodies (MoAb) recognizing neoepitopes exposed on activation products of complement proteins but hidden in the native components have been used for quantification of activated complement. A previously produced and characterized mouse MoAb, recognizing a neoepitope on the human plasma protein C1-inhibitor complexed with its substrates, was used to design an enzyme immunoassay for detection of C1-inhibitor complexed with C1r and C1s. These complexes are indicators of early classical complement pathway activation. The standard was serum activated with heat aggregated IgG defined to contain 1000 arbitrary units (AU)/ml. The lower detection limit was approximately 0.05 AU/ml corresponding to 0.005% of fully activated serum. The reliability of the assay, including day-to-day variation, was tested. Intra-assay variation coefficients were 12% for low plasma control and 13% for high plasma control (n = 12 for both). Inter-assay variation coefficients were 12% for low control (n = 6), 19% for high control (n = 6) and 15% for the normal plasma control (n = 9). A 2.5-97.5 percentile reference range (normal blood donors) was 16-33 AU/ml. Two patients with systemic lupus erythematosus had considerably elevated plasma levels of the activation product (56 and 62 AU/ml), and six patients with hereditary angioedema had normal plasma levels despite considerably reduced C1-inhibitor concentration. We conclude that the present method is sensitive and reliable for detection of early classical pathway activation and superior to previously published methods by utilizing neoepitope specificity and non-radiolabelled reagents.

Improved method for measuring C1-r-C1-s-(C1 inh)2 complexes by an enzyme-linked immunosorbent assay.

Measurement of C1-r-C1-s-(C1 inh)2 complexes in serum or plasma by enzyme-linked immunosorbent assay (ELISA) has been proposed as a relatively convenient and sensitive means for assessing C1 activation. However, interference by unactivated C1q (r-s)2 at low serum or plasma dilutions has resulted in estimates that vary widely with the degree of serum or plasma dilution. Precipitating the interfering C1q (r-s)2 with 6% polyethylene glycol has been proposed to resolve this problem, but here it is shown that this procedure also precipitates or coprecipitates some of the C1-r-C1-s-(C1 inh)2 complexes. Satisfactory results have been achieved without PEG precipitation by testing high plasma dilutions under conditions where there is a sufficient excess of anti-C1s coating the microtitration plate wells that removal of C1q (r-s)2 is not necessary. Optimizing conditions for quantitating these complexes at high dilution have been investigated. The mean normal EDTA plasma C1-r-C1-s-(C1 inh)2 complex measurement was 36.6 +/- 7.0 (S.D.) ELISA units with a 95% confidence interval of 19.5-47.6u. Besides providing a sensitive assay for C1 activation, measuring C1-r-C1-s-(C1 inh)2 complexes may help to clarify the pathophysiologic mechanisms resulting from C1 inh deficiency under various conditions.

Complement profiles in human skin lymph during the course of irritant contact dermatitis.

Using microsurgery a superficial peripheral lymph vessel draining the skin of the upper and medial part of the foot was cannulated on the lower leg of two healthy human volunteers. An irritant contact dermatitis was induced 2 days later by the application of 10% sodium lauryl sulphate to the drained skin area. After a further 3 days the spontaneously regressing skin reaction was treated with clobetasol propionate. Lymph was continuously collected in two aliquots per day for 7 days. The levels of total protein, of albumin and globulins, and of complement components of the classical, the alternative and the lytic pathway as well as the C4A and C4B gene products and the regulatory proteins FB, C1INH, C4BP, FH and FI were determined by ELISA and radial immunodiffusion techniques. Postoperatively, the levels of complement proteins and globulins in the lymph were 5-10 times lower than those in normal human serum, but increased during the course of the skin reaction, while the irritant contact dermatitis did not induce a change in their plasma concentration. In comparison to the baseline, the mean values for C1q, C1r, C2, C5, C6, C7, C8, C9, FB, C1INH, C4BP, FH and FI exhibited a 3-5-fold increase, C3, total C4, albumin and the alpha 1-globulin fraction a 6-9-fold increase, and C1s, C4A, C4B, FB and alpha 2-, beta- and gamma-globulins a 10-20-fold increase.(ABSTRACT TRUNCATED AT 250 WORDS)

Trimer and tetramer complexes containing C1 esterase inhibitor, C1r and C1s, in serum and synovial fluid of patients with rheumatic disease.

During activation, the first component of complement C1q (C1r-C1s)2 is dissociated in conjunction with the formation of complexes containing C1 esterase inhibitor (C1-INH). Trimer complexes, with zymogen C1s associated with a firm C1-INH-C1r complex (C1-INH-C1r-C1s) can be distinguished from tetramer complexes C1-INH-C1r-C1s-C1-INH) in which C1-INH is firmly bound to both proteases. In the present study a two-stage electroimmunoassay was developed for the specific measurement of C1-INH-C1r-C1s. In the first step, C1-INH and its complexes were immunoprecipitated with anti-C1-INH during electrophoresis in the presence of Ca2+. In the second step, C1s contained in C1-INH-C1r-C1s was dissociated in the presence of EDTA and was measured by immunoprecipitation with anti-C1s. C1-INH-C1r-C1s were consistently found in normal sera. Normal sera did not contain C1-INH-C1r-C1s-C1-INH as assessed with a previously described ELISA procedure. Sera and synovial fluids from two groups of patients with inflammatory arthritis were investigated. In rheumatoid arthritis patients (n = 15) C1-INH-C1r-C1s complexes were usually found at high concentration both in serum and synovial fluid. C1-INH-C1r-C1s-C1-INH complexes were also present with values that were higher in synovial fluid than in serum, in accord with previous findings of classical pathway activation in the inflamed joints of the patients. Patients with spondylarthritic syndromes (n = 7) had serum and synovial fluid C1-INH-C1r-C1s concentrations that were comparable to those of the rheumatoid arthritis patients. If at all present, C1-INH-C1r-C1s-C1-INH were detected in trace amounts. Thus, C1 activation in patients with spondylarthritic syndromes appeared to be efficiently controlled at the C1r level. Distinguishing between C1-INH-C1r-C1s and C1-INH-C1r-C1s-C1-INH may prove of value in further studies of the activation and control of C1 in disease.

An improved method for complement subcomponent C1R typing.

An improved method has been developed for the reliable classification of different C1R genetic variant forms from human serum or plasma. The method combines the use of neuraminidase-digested samples followed by monodimensional isoelectric focusing in the pH range 5 to 8 followed by immunoblotting. The method yields a simple pattern, with one major band in homozygote and two major bands in heterozygote cases.