Complement aberrations and autoantibodies to complement proteins in relation to disease mechanisms.

The complement system, a part of the innate immune system with several links to the adaptive immune system, plays an important role in the pathogenesis of many diseases. The purpose of this thesis was to document and clarify some of these mechanisms. The thesis is based on four papers (I-IV). (I and II) Autoantibodies to the C3 cleaving enzyme complex of the alternative pathway, C3 nephritic factors (C3 NeF), cause partial C3 deficiency and are associated with increased susceptibility to bacterial infections. By analysis of samples from 20 patients with C3 NeF, it was confirmed that C3 NeF are of at least 2 types; one with fluid phase (C3 NeF type I) and one with solid phase (C3 NeF type II) activity. Different types of C3 NeF were associated with different serum complement profiles and symptoms. Only C3 NeF type II were found to be associated with circulating autoantibodies to the collagenous region of C1q (aC1qCLR). Defense against Neisseria meningitidis in 26 patients with low C3 concentrations due to C3 NeF was investigated. In patients and control children, homozygosity for the IgG1 and IgG3 IGHG alleles G1M*f and G3M*b was found to be associated with higher serum bactericidal activity (SBA) than was heterozygosity. IGHG alleles correlated to IgG subclass binding to live meningococci, while IgG subclass binding did not correlate to SBA. Thus, the mechanism of influence from IGHG genes on SBA is unclear. IGHG variants are important for immune defense against meningococci in states of deficient complement function. This relationship should be examined further. (III) Serum levels of mannan-binding lectin (MBL) and antibodies to proteins from a potentially nephritogenic Streptococcus pyogenes strain (serotype M1 strain AP1) were investigated in 73 patients with acute poststreptococcal glomerulonephritis (AGN). Antibody responses to the serotype M1-related antigens M1, protein H and streptococcal inhibitor of complement were increased in patients compared to controls. The presence of MBL deficient individuals (serum concentration <0.1 mg/L) among AGN patients showed that the lectin pathway is not required in the pathogenesis of AGN. (IV) Autoimmunity has been implied to participate in the pathogenesis of idiopathic sudden hearing loss (ISHL). Autoantibodies were analysed in sera from 92 patients with ISHL. The most frequently occurring antibody was aC1qCLR, detected in 12 patients (13 %), all with normal serum concentrations of C1q. In ISHL, aC1qCLR probably represents cross-reactivity between C1q and inner ear protein(s)

Sensitive and specific assays for C3 nephritic factors clarify mechanisms underlying complement dysregulation

C3 nephritic factors are autoantibodies that prolong the half-life or prevent regulation of the alternative pathway C3 convertase, resulting in uncontrolled complement activation. They are strongly associated with renal disease but their role in pathogenesis remains controversial. Here we optimized and compared a panel of assays to identify and interrogate nephritic factor activities. Of 101 patients with histologic or clinically evident disease, 48 were positive in some or all assays. In the presence of properdin, binding of autoantibody was detected in 39 samples and convertase stabilization was detected in 36. Forty-two of 48 nephritic factors tested prevented convertase decay by factor H, and most of these by decay accelerating factor (28) and complement receptor 1 (34). Representative properdin-independent nephritic factors had no effect on C5 cleavage and terminal pathway activity, while properdin-dependent nephritic factors enhanced activity. Biacore analysis of four purified IgG samples confirmed resistance to decay and showed that properdin-independent nephritic factors increased convertase half-life over 50-fold, whereas properdin-dependent nephritic factors increased the half-life 10- to 20-fold and also increased activity of the C3 convertase up to 10-fold. Thus, our study provides a rational approach to detect and characterize nephritic factors in patients

Newborn screening for presymptomatic diagnosis of complement and phagocyte deficiencies

The clinical outcomes of primary immunodeficiencies (PIDs) are greatly improved by accurate diagnosis early in life. However, it is not common to consider PIDs before the manifestation of severe clinical symptoms. Including PIDs in the nation-wide newborn screening programs will potentially improve survival and provide better disease management and preventive care in PID patients. This calls for the detection of disease biomarkers in blood and the use of dried blood spot samples, which is a part of routine newborn screening programs worldwide. Here, we developed a newborn screening method based on multiplex protein profiling for parallel diagnosis of 22 innate immunodeficiencies affecting the complement system and respiratory burst function in phagocytosis. The proposed method uses a small fraction of eluted blood from dried blood spots and is applicable for population-scale performance. The diagnosis method is validated through a retrospective screening of immunodeficient patient samples. This diagnostic approach can pave the way for an earlier, more comprehensive and accurate diagnosis of complement and phagocytic disorders, which ultimately lead to a healthy and active life for the PID patientsThis work was supported by the Swedish Research Council (VR) and grants provided by the Stockholm County Council (ALF)

Clinical Complement Analysis—An Overview

The complement system plays an important role in varying types of disease, ranging from inflammatory and autoimmune disorders to immune deficiency states. In addition, new settings have emerged where complement analysis is of interest to monitor complement-directed therapy and aid identification of transplant complications. Therefore, it is critical that clinical laboratories offer optimized and timely complement analysis. This review presents a comprehensive overview of the most important complement analysis methods that are currently used. It also points to some areas within complement diagnostics where development is needed, for example, regarding certain analytes for which practical methods suitable for the routine laboratory are lacking. Furthermore, it contains a more detailed discussion on complement autoantibody assessment. The list of analyses providing clinically valuable information includes analysis of complement function, quantification of individual complement components and complement activation fragments, identification of autoantibodies to complement, as well as genetic complement analyses. There is still a shortage of commercially available methods suitable for high-throughput screening of complement deficiency and for assessment of complement activation, but development is under way. There is also ongoing work within the complement community to improve standardization of measurements, and recently, an extensive quality assurance program has been initiated

Hypocomplementaemia caused by C3 nephritic factors (C3 NeF): clinical findings and the coincidence of C3 NeF type II with anti-C1q autoantibodies

OBJECTIVES: The main purposes were to document manifestations associated with prolonged or clinically unexplained C3 deficiency and to approximate how often hypocomplementaemia of this kind is caused by C3 nephritic factors (C3 NeF), i.e. autoantibodies to alternative pathway C3 convertases. We also wished to distinguish between C3 NeF types I and II and to assess coincident autoantibody responses to the collagen-like region of C1q (C1qCLR). SETTING: The investigation was based on serum samples referred to a specialized laboratory for complement analysis in the course of several years. SUBJECTS: Twenty-five persons with C3 concentrations lower than 0.43 g L-1, a third of the normal, were included in the study. RESULTS: Analysis using three methods provided evidence of C3 NeF in 20 persons with equal frequencies of C3 NeF types I and II. We also gave evidence of antibody specificity differences for the two types of C3 NeF. Six patients with C3 NeF type II showed antibodies to C1qCLR. Membranoproliferative glomerulonephritis was the predominant diagnosis and two patients had partial lipodystrophy reflecting the well-known association between these diseases and C3 NeF. Anaphylactoid purpura, systemic lupus erythematosus, and severe infection, mainly meningococcal disease, were also observed. CONCLUSIONS: The study group was probably fairly representative of C3 deficiency syndromes as encountered in clinical practice. The findings emphasize the heterogeneity of C3 NeF, and that acquired C3 deficiency syndromes caused by C3 NeF should perhaps be considered more often in diagnostic work