Hemolytic Tests Exploring Factor H Functional Activities.

Impairment of the complement regulatory protein Factor H (FH) is implicated in the physiopathological mechanisms of different diseases like atypical hemolytic and uremic syndrome and C3 glomerulopathies. It may be due to genetic abnormalities or acquired with the development of autoantibodies. FH has several ligands; therefore, the exploration of its functions requires to perform different tests. Among them, two hemolytic tests are very useful because they give specific and complementary information about FH functions. The first one is dedicated to explore the FH capacity to dissociate the alternative pathway C3 convertase, whereas the second one is designed to explore the capacity of FH to bind cell surfaces and to protect them from complement attack. This chapter describes the procedures to perform these two hemolytic tests, exploring in a complementary way the FH functionality.

Detection of Autoantibodies to Complement Components by Surface Plasmon Resonance-Based Technology.

The innate immune complement system is a powerful defense cascade against pathogens, but can induce host tissue damage when overactivated. In pathological conditions, mainly but not restricted to renal diseases, such as lupus nephritis, atypical hemolytic uremic syndrome, and C3 glomerulopathies, complement is overactivated or dysregulated by autoantibodies directed against its components and regulators. Among the key autoantibody targets are the initiator of the classical complement pathway C1q, the alternative pathway regulator Factor H, the components of the alternative pathway C3 convertase complex C3 and Factor B and the convertase complex itself. This methodological article describes our experience with a method for detection of anti-complement autoantibodies in real time using surface plasmon resonance-based technology. It allows label-free evaluation of the binding efficacy and stability of the formed antigen-antibody complexes.

Gastrointestinal pathogens in anti-FH antibody positive and negative Hemolytic Uremic Syndrome.

BACKGROUND: Prodromal symptoms are frequently reported in the atypical form of Hemolytic uremic syndrome (aHUS) suggesting implication of infectious triggers. Some pathogens may also play a role in the mechanisms of production of autoantibody directed against Factor H (FH), a complement regulator, leading to aHUS. METHODS: The presence of 15 gastrointestinal (GI) pathogens was investigated by using xTAG-based multiplex PCR techniques on stools collected at the acute phase in a cohort of Indian HUS children classified according to the presence or absence of anti-FH autoantibodies. RESULTS: Prevalence of pathogens in patients with anti-FH antibody (62.5%) was twice that in those without (31.5%). Different pathogens were detected, the most frequent being Clostridium difficile, Giardia intestinalis, Salmonella, Shigella, Rotavirus, Norovirus and Entamoeba histolytica. No stool was positive for Shigatoxin. CONCLUSION: This study reveals a higher prevalence of GI pathogens in anti-FH positive than in negative patients. No single pathogen was implicated exclusively in one form of HUS. These pathogens may play a role in the disease initiation by inducing complement activation or an autoimmune response.

A novel CFHR1-CFHR5 hybrid leads to a familial dominant C3 glomerulopathy.

The intrinsic similarity shared between the members of the complement factor H family, which comprises complement factor H and five complement factor H-related (CFHR) genes, leads to various recombination events. In turn these events lead to deletions of some genes or abnormal proteins, which are found in patients with atypical hemolytic uremic syndrome or C3 glomerulopathies. Here we describe a novel genetic rearrangement generated from a heterozygous deletion spanning 146 Kbp involving multiple CFHR genes leading to a CFHR1-R5 hybrid protein. This deletion was found in four family members presenting with a familial dominant glomerulopathy histologically classified as an overlap of dense deposit disease and C3 glomerulonephritis. Affected patients exhibited permanently low C3 and factor B levels and high amounts of activation fragments sC5b9 and Bb, indicating a systemic alternative pathway dysregulation. The abnormal protein, characterized by Western blot and immunoprecipitation, was shown to circulate in association with CFHR1 and CFHR2, attributable to its two N-terminal dimerization motifs. The presence of this protein is associated with a perturbation of Factor H activity on the C3 convertase decay. Thus, our study highlights the role of CFHRs in the physiopathology of C3 glomerulopathies and stresses the importance of screening CFHRs in all familial C3 glomerulopathies. Such hybrids described till now were always associated with familial forms.