Anti-Ficolin-2 and Anti-Ficolin-3 Autoantibody Detection by ELISA

International audienceFicolins are recognition proteins of the lectin pathway of the complement system and also play an important role in innate immunity and in the maintenance of tissue homeostasis. They deserve special attention in the context of autoimmunity since they are involved in the uptake of dying cells. Because the monitoring of systemic lupus erythematosus (SLE) patients is particularly difficult, it is crucial to find new relevant serum biomarkers. The ability to detect autoantibodies in the patients' sera provides a diagnostic and prognostic advantage. We describe in this chapter quantitative enzyme linked immunosorbent assays (ELISA) to detect the presence of autoantibodies targeting ficolin-2 and ficolin-3 in human sera. Recombinant ficolins produced in a mammalian expression system are used as coating antigens. The described in-house ELISAs provide a valuable tool to efficiently quantify anti-ficolin autoantibodies in the sera of SLE patients

Involvement of complement pathways in patients with bacterial septicemia.

The complement system is a major humoral portion of the innate immune system, playing a significant role in host defence against microorganisms. The biological importance of this system is underlined by the fact that at least three different pathways for its activation exist, the classical, the MBL and the alternative pathway. To elucidate the involvement of the classical and/or the MBL pathway during bacterial septicemia, 32 patients with gram-positive and 30 patients with gram-negative bacterial infections were investigated. In patients with gram-positive bacteria, a significant consumption of C1q (p=0.005) but not of mannose-binding lectin (MBL) (p=0.2) was found during the acute phase of infection. In contrast, in patients with gram-negative bacterial infections, a significant reduction of MBL (p=0.002) and only a moderate, less significant reduction of C1q (p=0.03) were observed. As a model for the binding of MBL to gram-negative bacteria, Salmonella strains with defined mutations in their lipopolysaccharide (LPS) structure were used. The comparison of the binding of MBL to these Salmonella strains with that of the corresponding isolated LPS forms bound to microtiter plates revealed a similar binding pattern, supporting the interpretation that LPS on the surface of gram-negative bacteria is the major acceptor molecule for MBL on these bacteria, which according to our results obviously also takes place during gram-negative bacterial septicaemia. Furthermore, we were able to demonstrate that MBL bound to LPS was able to initiate activation of the complement cascade as measured by the occurrence of the cleavage product C4c

Complement System and Alarmin HMGB1 Crosstalk: For Better or Worse

International audienceOur immune system responds to infectious (PAMPs) and tissue damage (DAMPs) signals. The complement system and alarmin High-Mobility Group Box 1 (HMGB1) are two powerful soluble actors of human host defense and immune surveillance. These systems involve molecular cascades and amplification loops for their signaling or activation. Initially activated as alarm raising systems, their function can be finally switched towards inflammation resolution, where they sustain immune maturation and orchestrate repair mechanisms, opening the way back to homeostasis. However, when getting out of control, these defense systems can become deleterious and trigger serious cellular and tissue damage. Therefore, they can be considered as double-edged swords. The close interaction between the complement and HMGB1 pathways is described here, as well as their traditional and non-canonical roles, their functioning at different locations and their independent and collective impact in different systems both in health and disease. Starting from these systems and interplay at the molecular level (when elucidated), we then provide disease examples to better illustrate the signs and consequences of their roles and interaction, highlighting their importance and possible vicious circles in alarm raising and inflammation, both individually or in combination. Although this integrated view may open new therapeutic strategies, future challenges have to be faced because of the remaining unknowns regarding the molecular mechanisms underlying the fragile molecular balance which can drift towards disease or return to homeostasis, as briefly discussed at the end

Towards a specific marker for acute bradykinin-mediated angioedema attacks: a literature review.

International audienceBradykinin-mediated angioedema (AE) is a rare disease characterised by recurrent angioedema linked to acquired (e.g. angiotensin converting enzyme inhibitor induced AE) or hereditary disorders (e.g. AE type I or II). As the clinical picture can be misleading, diagnosis of this disease is sometimes difficult. A bradykinin-mediated AE attack may be a therapeutic emergency which requires access to effective, but expensive, treatments. Their prescription must therefore be justified. No specific marker of acute bradykinin-mediated AE attacks has yet been identified to facilitate the therapeutic decision but it has been sought in many studies

The Immunopathology of Complement Proteins and Innate Immunity in Autoimmune Disease

International audienceThe complement is a powerful cascade of the innate immunity and also acts as a bridge between innate and acquired immune defence. Complement activation can occur via three distinct pathways, the classical, alternative and lectin pathways, each resulting in the common terminal pathway. Complement activation results in the release of a range of biologically active molecules that significantly contribute to immune surveillance and tissue homeostasis. Several soluble and membrane-bound regulatory proteins restrict complement activation in order to prevent complement-mediated autologous damage, consumption and exacerbated inflammation. The crucial role of complement in the host homeostasis is illustrated by association of both complement deficiency and overactivation with severe and life-threatening diseases. Autoantibodies targeting complement components have been described to alter expression and/or function of target protein resulting in a dysregulation of the delicate equilibrium between activation and inhibition of complement. The spectrum of diseases associated with complement autoantibodies depends on which complement protein and activation pathway are targeted, ranging from autoimmune disorders to kidney and vascular diseases. Nevertheless, these autoantibodies have been identified as differential biomarkers for diagnosis or follow-up of disease only in a small number of clinical conditions. For some autoantibodies, a clear relationship with clinical manifestations has been identified, such as anti-C1q, anti-Factor H, anti-C1 Inhibitor antibodies and C3 nephritic factor. For other autoantibodies, the origin and the functional consequences still remain to be elucidated, questioning about the pathophysiological significance of these autoantibodies, such as anti-mannose binding lectin, anti-Factor I, anti-Factor B and anti-C3b antibodies. The detection of autoantibodies targeting complement components is performed in specialized laboratories; however, there is no consensus on detection methods and standardization of the assays is a real challenge. This review summarizes the current panorama of autoantibodies targeting complement recognition proteins of the classical and lectin pathways, associated proteases, convertases, regulators and terminal components, with an emphasis on autoantibodies clearly involved in clinical conditions

Bulbing in long-day onion (Allium cepa L.) cultured in vitro: Comparison between sugar feeding and light induction

Bulbs were obtained on onion plants cultured in vitro. No bulbing happened under long days with fluorescent light and 30–40 g l−1 sucrose. Bulb formation was mainly dependent either on sucrose concentration in the culture medium, or on light spectral composition. Raising the sucrose concentration from 40 to 120 g l−1 increased plant basal swelling and stopped further vegetative development. These plants were not dormant. When fluorescent light was enriched in incandescence during a long day period, bulbs were obtained in two months and underwent a consecutive dormancy. (Résumé d'auteur

Antibodies targeting circulating protective molecules in lupus nephritis: Interest as serological biomarkers

International audienceLupus nephritis (LN) is one of the most frequent and severe manifestations of systemic lupus erythematosus (SLE), considered as the major predictor of poor prognosis. An early diagnosis of LN is a real challenge in the management of SLE and has an important implication in guiding treatments. In clinical practice, conventional parameters still lack sensitivity and specificity for detecting ongoing disease activity in lupus kidneys and early relapse of nephritis. LN is characterized by glomerular kidney injury, essentially due to deposition of immune complexes involving autoantibodies against cellular components and circulating proteins. One of the possible mechanisms of induction of autoantibodies in SLE is a defect in apoptotic cells clearance and subsequent release of intracellular autoantigens. Autoantibodies against soluble protective molecules involved in the uptake of dying cells, including complement proteins and pentraxins, have been described. In this review, we present the main autoantibodies found in LN, with a focus on the antibodies against these protective molecules. We also discuss their pathogenic role and conclude with their potential interest as serological biomarkers in LN

Prévalence et caractéristiques de l’auto-immunité sérique chez les patientes suivies pour infertilité au CHU de Grenoble

International audienceFertility disorders in autoimmune diseases are well described. However, little is known about the presence of a humoral serum autoimmunity in case of infertility (antinuclear antibodies, ACAN or antiphospholipid, APL) without criteria of autoimmune disease

Effects of immunoadsorption combined with membrane filtration on complement markers – results of a randomized, controlled, crossover study

International audienceThe complement system has been implicated in several kidney diseases, such as antibody-mediated rejection after kidney transplantation. Antibody-depletion techniques allow successful ABO- and/or HLA-incompatible transplantation. Considering the IgG removal, the use of semi-selective immunoadsorption (IA) has been advocated. However, because of results on incomplete IgM depletion, the adjunctive use of membrane filtration (MF) has been proposed to enhance the removal of macromolecules and to interfere with complement activation. This secondary endpoint analysis of a recently published randomized, controlled, cross-over trial was designed to investigate the effect of combined treatment IA + MF compared to IA alone on complement depletion. Two treatment sequences, a single session of IA + MF followed by IA (and vice versa), were analyzed with regard to C5b-9, properdin, and mannose-binding lectin (MBL) levels. Neither IA alone nor IA + MF provoked complement activation as demonstrated by stable low levels of C5b-9 after the procedure as compared to the previous. The combined treatment substantially lowered properdin (77% vs. 26% reduction, P < 0.0001) as well as MBL concentrations (81% vs. 11% reduction, P < 0.0001). Recovery of properdin and MBL levels appears to be longer after IA alone compared to IA + MF. Depletion of properdin and MBL levels may have potential clinical implications in the setting of kidney transplantation