Human complement factor H

We isolated cDNA clones coding for the functionally important tryptic N-terminal38- kDa fragment of human complement control protein factor H using polyclonal and monoclonal antibodies to screen a human liver cDNA library cloned in a bacterial expression vector, PEX-1. By testing the reactivity of antibodies specific for the recombinant proteins produced by individual clones with proteolytic fragments of serum H the exact position of these cDNA clones within H was mapped. One clone, H-19, coding for the 38-kDa fragment of H was sequenced and found to code for 289 amino acids derived from the 38-kDa N-terminal fragment as well as for the first 108 amino acids belonging to the complementary 142-kDa tryptic fragment. The derived protein sequence could be arranged in 6 highly homologous repeats of about 60 amino acids each, the homology between the repeats being determined by the characteristic position of cysteine, proline, glycine, tyrosine and tryptophane residues. The region coding for the epitope recognized by one of our monoclonal antibodies was localized by subcloning restriction fragments of H-19 into the expression plasmid and testing for the expression of this epitope

Complement activation and protein adsorption by carbon nanotubes

As a first step to validate the use of carbon nanotubes as novel vaccine or drug delivery devices, their interaction with a part of the human immune system, complement, has been explored. Haemolytic assays were conducted to investigate the activation of the human serum complement system via the classical and alternative pathways. Western blot and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) techniques were used to elucidate the mechanism of activation of complement via the classical pathway, and to analyse the interaction of complement and other plasma proteins with carbon nanotubes. We report for the first time that carbon nanotubes activate human complement via both classical and alternative pathways. We conclude that complement activation by nanotubes is consistent with reported adjuvant effects, and might also in various circumstances promote damaging effects of excessive complement activation, such as inflammation and granuloma formation. C1q binds directly to carbon nanotubes. Protein binding to carbon nanotubes is highly selective, since out of the many different proteins in plasma, very few bind to the carbon nanotubes. Fibrinogen and apolipoproteins (AI, AIV and CIII) were the proteins that bound to carbon nanotubes in greatest quantit

Hyperfunctional complement C3 promotes C5-dependent atypical hemolytic uremic syndrome in mice

Atypical hemolytic uremic syndrome (aHUS) is frequently associated in humans with loss-of-function mutations in complement-regulating proteins or gain-of-function mutations in complement-activating proteins. Thus, aHUS provides an archetypal complement-mediated disease with which to model new therapeutic strategies and treatments. Herein, we show that, when transferred to mice, an aHUS-associated gain-of-function change (D1115N) to the complement-activation protein C3 results in aHUS. Homozygous C3 p.D1115N (C3KI) mice developed spontaneous chronic thrombotic microangiopathy together with hematuria, thrombocytopenia, elevated creatinine, and evidence of hemolysis. Mice with active disease had reduced plasma C3 with C3 fragment and C9 deposition within the kidney. Therapeutic blockade or genetic deletion of C5, a protein downstream of C3 in the complement cascade, protected homozygous C3KI mice from thrombotic microangiopathy and aHUS. Thus, our data provide in vivo modeling evidence that gain-of-function changes in complement C3 drive aHUS. They also show that long-term C5 deficiency is not accompanied by development of other renal complications (such as C3 glomerulopathy) despite sustained dysregulation of C3. Our results suggest that this preclinical model will allow testing of novel complement inhibitors with the aim of developing precisely targeted therapeutics that could have application in many complement-mediated diseases.</p