Low-molecular weight inhibitors of the alternative complement pathway.

Dysregulation of the alternative complement pathway predisposes individuals to a number of diseases. It can either be evoked by genetic alterations in or by stabilizing antibodies to important pathway components and typically leads to severe diseases such as paroxysmal nocturnal hemoglobinuria, atypical hemolytic uremic syndrome, C3 glomerulopathy, and age-related macular degeneration. In addition, the alternative pathway may also be involved in many other diseases where its amplifying function for all complement pathways might play a role. To identify specific alternative pathway inhibitors that qualify as therapeutics for these diseases, drug discovery efforts have focused on the two central proteases of the pathway, factor B and factor D. Although drug discovery has been challenging for a number of reasons, potent and selective low-molecular weight (LMW) oral inhibitors have now been discovered for both proteases and several molecules are in clinical development for multiple complement-mediated diseases. While the clinical development of these inhibitors initially focuses on diseases with systemic and/or peripheral tissue complement activation, the availability of LMW inhibitors may also open up the prospect of inhibiting complement in the central nervous system where its activation may also play an important role in several neurodegenerative diseases.

An intact zinc finger motif of the C1B domain is critical for stability and activity of diacylglycerol kinase-ε.

Diacylglycerol kinase-ε (DGKε) phosphorylates DAG to phosphatidic acid with unique specificity toward 18:0/20:4 DAG (SAG). SAG is a typical backbone of phosphatidylinositol and its derivatives, therefore DGKε activity is crucial for the turnover of these signaling lipids. Malfunction of DGKε contributes to several pathophysiological conditions, including atypical hemolytic uremic syndrome (aHUS) linked with DGKE mutations. In the present study we analyzed the role of a zinc finger motif of the C1B domain of DGKε, as some aHUS-linked mutations affect this ill-defined part of the kinase. For this, we introduce a novel fluorescent assay for determination of DGKε activity which relies on the use of NBD-SAG in mixed micelles as a substrate, followed by TLC separation of NBD-phosphatidic acid formed. The assay reliably determines the activity of purified human GST-DGKε, also endogenous DGKε or overexpressed mouse DGKε-Myc in cell lysates, homogenates, and kinase immunoprecipitates. Using the above assay we found that four amino acids, Cys135, Cys138, His161 and Cys164, forming the zinc finger motif in the C1B domain are required for the DGKε-Myc activity and stability. Substitution of any of these amino acids with Ala or Trp in DGKε-Myc abolished its activity and led to its proteasomal degradation, possibly assisted by Hsp70/90/40 chaperones. Inhibition of the 26S proteasome prevented the degradation but the mutated proteins were inactive. The present data on the deleterious effect of the zinc finger motif disruption contribute to the understanding of the DGKε-linked aHUS, as the Cys164Trp substitution in mouse DGKε corresponds to the Cys167Trp one in human DGKε found in some aHUS patients.

Antibody recognition of complement factor H reveals a flexible loop involved in atypical hemolytic uremic syndrome pathogenesis.

Atypical hemolytic uremic syndrome (aHUS) is a disease associated with dysregulation of the immune complement system, especially of the alternative pathway (AP). Complement factor H (CFH), consisting of 20 domains called complement control protein (CCP1-20), downregulates the AP as a cofactor for mediating C3 inactivation by complement factor I. However, anomalies related to CFH are known to cause excessive complement activation and cytotoxicity. In aHUS, mutations and the presence of anti-CFH autoantibodies (AAbs) have been reported as plausible causes of CFH dysfunction, and it is known that CFH-related aHUS carries a high probability of end-stage renal disease. Elucidating the detailed functions of CFH at the molecular level will help to understand aHUS pathogenesis. Herein, we used biophysical data to reveal that a heavy-chain antibody fragment, termed VHH4, recognized CFH with high affinity. Hemolytic assays also indicated that VHH4 disrupted the protective function of CFH on sheep erythrocytes. Furthermore, X-ray crystallography revealed that VHH4 recognized the Leu1181-Leu1189CCP20 loop, a known anti-CFH AAbs epitope. We next analyzed the dynamics of the C-terminal region of CFH and showed that the epitopes recognized by anti-CFH AAbs and VHH4 were the most flexible regions in CCP18-20. Finally, we conducted mutation analyses to elucidate the mechanism of VHH4 recognition of CFH and revealed that VHH4 inserts the Trp1183CCP20 residue of CFH into the pocket formed by the complementary determining region 3 loop. These results suggested that anti-CFH AAbs may adopt a similar molecular mechanism to recognize the flexible loop of Leu1181-Leu1189CCP20, leading to aHUS pathogenesis.

Atypical Hemolytic Uremic Syndrome-Associated FHR1 Isoform FHR1*B Enhances Complement Activation and Inflammation.

Atypical hemolytic uremic syndrome (aHUS) is a rare but severe type of thrombotic microangiopathy that is triggered by the abnormal activation of the alternative complement pathway. Previous studies have reported that three completely linked coding variants of CFHR1 form two haplotypes, namely, CFHR1*A (c.469C, c.475C, c.523G) and CFHR1*B (c.469T, c.475G, c.523C). CFHR1*B is associated with susceptibility to aHUS. To explore the genetic mechanism by which CFHR1 isoforms contribute to aHUS, we compared the structures of FHR1*A and FHR1*B by homology modeling and found differences in the angles between SCR3 and SCR4-SCR5, as FHR1*B had a larger angle than FHR1*A. Then, we expressed FHR1*A and FHR1*B recombinant proteins and compared their functions in complement system regulation and inflammation. We found that FHR1*B presented a significantly higher capacity for binding C3b and necrotic cells than FHR1*A. In a cofactor assay, the FHR-1*B showed stronger influence on FH mediated cofactor function than the FHR-1*A, resulted in fewer C3b cleavage products. In the C3 convertase assays, FHR1*B showed more powerful effect compared with FHR1*A regarding to de-regulate FH function of inhibition the assembling of C3bBb. Additionally, we also found that FHR1*B triggered monocytes to secrete higher levels of IL-1ß and IL-6 than FHR1*A. In the present study, we showed that variants of CFHR1 might differently affect complement activation and sterile inflammation. Our findings provide a possible mechanism underlying the predisposition to aHUS caused by CFHR1 isoform CFHR1*B.

Patient-specific iPSC-derived endothelial cells reveal aberrant p38 MAPK signaling in atypical hemolytic uremic syndrome.

Atypical hemolytic uremic syndrome (aHUS) is a rare disease associated with high morbidity and mortality. Existing evidence suggests that the central pathogenesis to aHUS might be endothelial cell damage. Nevertheless, the role of endothelial cell alterations in aHUS has not been well characterized and the underlying mechanisms remain unclear. Utilizing an induced pluripotent stem cell-derived endothelial cell (iPSC-EC) model, we showed that anti-complement factor H autoantibody-associated aHUS patient-specific iPSC-ECs exhibited an intrinsic defect in endothelial functions. Stimulation using aHUS serums exacerbated endothelial dysfunctions, leading to cell apoptosis in iPSC-ECs. Importantly, we identified p38 as a novel signaling pathway contributing to endothelial dysfunctions in aHUS. These results illustrate that iPSC-ECs can be a reliable model to recapitulate EC pathological features, thus providing a unique platform for gaining mechanistic insights into EC injury in aHUS. Our findings highlight that the p38 MAPK signaling pathway can be a therapeutic target for treatment of aHUS.

Functional characterization of 105 factor H variants associated with aHUS: lessons for variant classification.

Atypical hemolytic uremic syndrome (aHUS) is a life-threatening thrombotic microangiopathy that can progress, when untreated, to end-stage renal disease. Most frequently, aHUS is caused by complement dysregulation due to pathogenic variants in genes that encode complement components and regulators. Among these genes, the factor H (FH) gene, CFH, presents with the highest frequency (15% to 20%) of variants and is associated with the poorest prognosis. Correct classification of CFH variants as pathogenic or benign is essential to clinical care but remains challenging owing to the dearth of functional studies. As a result, significant numbers of variants are reported as variants of uncertain significance. To address this knowledge gap, we expressed and functionally characterized 105 aHUS-associated FH variants. All FH variants were categorized as pathogenic or benign and, for each, we fully documented the nature of the pathogenicity. Twenty-six previously characterized FH variants were used as controls to validate and confirm the robustness of the functional assays used. Of the remaining 79 uncharacterized variants, only 29 (36.7%) alter FH expression or function in vitro and, therefore, are proposed to be pathogenic. We show that rarity in control databases is not informative for variant classification, and we identify important limitations in applying prediction algorithms to FH variants. Based on structural and functional data, we suggest ways to circumvent these difficulties and, thereby, improve variant classification. Our work highlights the need for functional assays to interpret FH variants accurately if clinical care of patients with aHUS is to be individualized and optimized.

Loss of diacylglycerol kinase ε causes thrombotic microangiopathy by impairing endothelial VEGFA signaling.

Loss of function of the lipid kinase diacylglycerol kinase ε (DGKε), encoded by the gene DGKE, causes a form of atypical hemolytic uremic syndrome that is not related to abnormalities of the alternative pathway of the complement, by mechanisms that are not understood. By generating a potentially novel endothelial specific Dgke-knockout mouse, we demonstrate that loss of Dgke in the endothelium results in impaired signaling downstream of VEGFR2 due to cellular shortage of phosphatidylinositol 4,5-biphosphate. Mechanistically, we found that, in the absence of DGKε in the endothelium, Akt fails to be activated upon VEGFR2 stimulation, resulting in defective induction of the enzyme cyclooxygenase 2 and production of prostaglandin E2 (PGE2). Treating the endothelial specific Dgke-knockout mice with a stable PGE2 analog was sufficient to reverse the clinical manifestations of thrombotic microangiopathy and proteinuria, possibly by suppressing the expression of matrix metalloproteinase 2 through PGE2-dependent upregulation of the chemokine receptor CXCR4. Our study reveals a complex array of autocrine signaling events downstream of VEGFR2 that are mediated by PGE2, that control endothelial activation and thrombogenic state, and that result in abnormalities of the glomerular filtration barrier.

The Benefits of Complement Measurements for the Clinical Practice.

The complement cascade is an evolutionary ancient innate immune defense system, playing a major role in the defense against infections. Its function in maintaining host homeostasis on activated cells has been emphasized by the crucial role of its overactivation in ever growing number of diseases, such as atypical hemolytic uremic syndrome (aHUS), autoimmune diseases as systemic lupus erythematosus (SLE), C3 glomerulopathies (C3GN), age-related macular degeneration (AMD), graft rejection, Alzheimer disease, and cancer, to name just a few. The last decade of research on complement has extended its implication in many pathological processes, offering new insights to potential therapeutic targets and asserting the necessity of reliable, sensitive, specific, accurate, and reproducible biomarkers to decipher complement role in pathology. We need to evaluate accurately which pathway or role should be targeted pharmacologically, and optimize treatment efficacy versus toxicity. This chapter is an introduction to the role of complement in human diseases and the use of complement-related biomarkers in the clinical practice. It is a part of a book intending to give reliable and standardized methods to evaluate complement according to nowadays needs and knowledge.

Ravulizumab: A Review in Atypical Haemolytic Uraemic Syndrome.

Ravulizumab (Ultomiris®), a humanized monoclonal antibody that inhibits complement protein C5, is indicated for the treatment of atypical haemolytic uraemic syndrome (aHUS) in several countries, including the USA and those of the EU. Ravulizumab has been re-engineered from eculizumab to extend its terminal elimination half-life, resulting in a more convenient maintenance dosage regimen of once every 4-8 weeks compared with once every 2-3 weeks for eculizumab. In single-arm phase 3 trials, ravulizumab resolved thrombotic microangiopathy in 54% and 78% of treatment-naïve adult and paediatric patients with aHUS, respectively, within 26 weeks. Ravulizumab was also effective in patients with postpartum aHUS and paediatric patients who responded to eculizumab and later switched to ravulizumab. Ravulizumab was generally well tolerated, with no unexpected safety events. The most common treatment-related adverse events with ravulizumab in treatment-naïve patients include headache, diarrhoea and vomiting. With its convenient once every 4-8 weeks maintenance regimen, ravulizumab is an important treatment option for aHUS in adult and paediatric patients.

Eculizumab discontinuation in children and adults with atypical hemolytic-uremic syndrome: a prospective multicenter study.

The optimal duration of eculizumab treatment in patients with atypical hemolytic uremic syndrome (aHUS) remains poorly defined. We conducted a prospective national multicenter open-label study to assess eculizumab discontinuation in children and adults with aHUS. Fifty-five patients (including 19 children) discontinued eculizumab (mean treatment duration, 16.5 months). Twenty-eight patients (51%) had rare variants in complement genes, mostly in MCP (n = 12; 22%), CFH (n = 6; 11%), and CFI (n = 6; 10%). At eculizumab discontinuation, 17 (30%) and 4 patients (7%) had stage 3 and 4 chronic kidney disease, respectively. During follow-up, 13 patients (23%; 6 children and 7 adults) experienced aHUS relapse. In multivariable analysis, female sex and presence of a rare variant in a complement gene were associated with an increased risk of aHUS relapse, whereas requirement for dialysis during a previous episode of acute aHUS was not. In addition, increased sC5b-9 plasma level at eculizumab discontinuation was associated with a higher risk of aHUS relapse in all patients and in the subset of carriers with a complement gene rare variant, both by log-rank test and in multivariable analysis. Of the 13 relapsing patients, all of whom restarted eculizumab, 11 regained their baseline renal function and 2 had a worsening of their preexisting chronic kidney disease, including 1 patient who progressed to end-stage renal disease. A strategy of eculizumab discontinuation in aHUS patients based on complement genetics is reasonable and safe. It improves the management and quality of life of a sizeable proportion of aHUS patients while reducing the cost of treatment. This trial was registered at www.clinicaltrials.gov as #NCT02574403.

Late renal recovery after treatment over 1 year post-onset in an atypical hemolytic uremic syndrome: a case report.

BACKGROUND: Atypical hemolytic uremic syndrome (aHUS) is a life-threatening disease that leads to end-stage kidney disease if only a poor response to plasma exchanges (PEs) or eculizumab therapy is achieved. CASE PRESENTATION: A 58-year-old Japanese man presented with thrombocytopenia, anemia, and kidney failure requiring dialysis without any underlying disease. A kidney biopsy revealed marked mesangiolysis in all glomeruli, compatible with thrombotic microangiopathy (TMA). Based on the positive anti- factor H antibody and negative result for secondary TMA, we diagnosed him as aHUS. Despite eculizumab administration after eight sessions of PE, neither platelet normalization nor kidney recovery was achieved. Eight months later, we discontinued eculizumab therapy due to anaphylactic reaction. At 15 months after the onset of TMA, his platelet count increased gradually from 40 to 150 × 103/µL with a decreased serum creatinine level and increased urine output, eventually allowing the withdrawal of dialysis therapy. A second kidney biopsy showed mesangial widening compatible with the healing of TMA. CONCLUSIONS: This case indicates that aHUS with PEs and eculizumab therapy has the potential for renal recovery even if over 1 year has passed.

Discovery of 4-((2S,4S)-4-Ethoxy-1-((5-methoxy-7-methyl-1H-indol-4-yl)methyl)piperidin-2-yl)benzoic Acid (LNP023), a Factor B Inhibitor Specifically Designed To Be Applicable to Treating a Diverse Array of Complement Mediated Diseases.

The alternative pathway (AP) of the complement system is a key contributor to the pathogenesis of several human diseases including age-related macular degeneration, paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic uremic syndrome (aHUS), and various glomerular diseases. The serine protease factor B (FB) is a key node in the AP and is integral to the formation of C3 and C5 convertase. Despite the prominent role of FB in the AP, selective orally bioavailable inhibitors, beyond our own efforts, have not been reported previously. Herein we describe in more detail our efforts to identify FB inhibitors by high-throughput screening (HTS) and leveraging insights from several X-ray cocrystal structures during optimization efforts. This work culminated in the discovery of LNP023 (41), which is currently being evaluated clinically in several diverse AP mediated indications.

Case Report: Variable Pharmacokinetic Profile of Eculizumab in an aHUS Patient.

Background: With the introduction of eculizumab, a C5-inhibitor, morbidity and mortality improved significantly for patients with atypical hemolytic uremic syndrome (aHUS). In view of the high costs, actual needs of the drug, and increasing evidence in literature, aHUS patients can be treated according to a restrictive eculizumab regimen. We retrospectively analyzed the pharmacokinetic and dynamic parameters of eculizumab in one patient in time, emphasizing various factors which could be taken into account during tapering of treatment. Case Presentation: A nowadays 18-year-old male with a severe, frequently relapsing form of atypical HUS due to a hybrid CFH/CFHR1 gene in combination with the homozygous factor H haplotype, required chronic plasma therapy (PT), including periods with plasma infusion, from the age of onset at 5 months until initiation of eculizumab at the age of 11 years. A mild but stable chronic kidney disease (CKD) and 9 years of disease remission enabled prolongation of eculizumab interval. At the age of 15 years, a sudden yet multifactorial progression of chronic kidney disease (CKD) was observed, without any signs of disease recurrence. However, an acquired glomerulocystic disease, a reduced left kidney function, and abnormal abdominal venous system of unknown etiology were found. In addition, after an aHUS relapse, an unexpected increase in intra-patient variability of eculizumab concentrations was seen. Retrospective pharmacokinetic analysis revealed a change in eculizumab clearance, associated with a simultaneous increase in proteinuria. Conclusion: High intra-patient variability of eculizumab pharmacokinetics were observed over time, emphasizing the necessity for adequate and continuous therapeutic drug monitoring in aHUS patients. Eculizumab serum trough levels together with complement activation markers (CH50) should be frequently assessed, especially during tapering of drug therapy and/or changing clinical conditions in the patient. In addition, an increase in proteinuria could result in urinary eculizumab loss, indicating that urinary monitoring of eculizumab may be important in aHUS patients with an unexplained decline in serum concentrations.

Three months interval therapy of Eculizumab in a patient with atypical hemolytic uremic syndrome with hybrid CFHR1/CFH gene.

Pregnancy-associated atypical hemolytic uremic syndrome (P-aHUS) is a rare condition. It is characterized by very high maternal mortality and morbidity. Most cases of P-aHUS (79%) manifest in the postpartum period; this is probably due to the complement's involvement in aHUS pathogenesis. Eculizumab is approved for aHUS treatment, but its use is limited due to cost, unknown duration of treatment, and vague dose intervals to keep patients in remission. In this case report, we present a 26-year-old female with P-aHUS with hybrid CFHR1/CFH gene. Eculizumab was initiated after 5 weeks of being on hemodialysis and plasmapheresis sessions. Full remission successfully achieved after 6th dose of Eculizumab, within 13 weeks of onset of aHUS. Due to financial issues and inability to financially cover the cost, Eculizumab was set in hold. Within 6 months, she suffered recurrence of the disease and Eculizumab was re-instated. After re-inducing full remission, the patient was switched to Eculizumab every 3 months instead of the recommended manufacture dose interval of every 2 weeks. We followed this patient for 3 years and she continued to be in remission based on clinical and laboratory data. In conclusion, achievement of successful and maintenance of remission of P-aHUS in this patient who had limited access to Eculizumab raise the attention of the efficacy of Eculizumab at longer time intervals. However, it is time to consider conducting a long-term study to learn about the safety and efficacy of this approach, which may have a major financial advantage for patients.

Genotype-phenotype correlations of low-frequency variants in the complement system in renal disease and age-related macular degeneration.

Genetic alterations in the complement system have been linked to a variety of diseases, including atypical hemolytic uremic syndrome (aHUS), C3 glomerulopathy (C3G), and age-related macular degeneration (AMD). We performed sequence analysis of the complement genes complement factor H (CFH), complement factor I (CFI), and complement C3 (C3) in 866 aHUS/C3G and 697 AMD patients. In total, we identified 505 low-frequency alleles, representing 121 unique variants, of which 51 are novel. CFH contained the largest number of unique low-frequency variants (n = 64; 53%), followed by C3 (n = 32; 26%) and CFI (n = 25; 21%). A substantial number of variants were found in both patients groups (n = 48; 40%), while 41 (34%) variants were found only in aHUS/C3G and 32 (26%) variants were AMD specific. Genotype-phenotype correlations between the disease groups identified a higher frequency of protein altering alleles in short consensus repeat 20 (SCR20) of factor H (FH), and in the serine protease domain of factor I (FI) in aHUS/C3G patients. In AMD, a higher frequency of protein-altering alleles was observed in SCR3, SCR5, and SCR7 of FH, the SRCR domain of FI, and in the MG3 domain of C3. In conclusion, we observed a substantial overlap of variants between aHUS/C3G and AMD; however, there is a distinct clustering of variants within specific domains.

Brain microvascular endothelial cells exhibit lower activation of the alternative complement pathway than glomerular microvascular endothelial cells.

Atypical hemolytic uremic syndrome (aHUS) and bone marrow transplantation-associated thrombotic microangiopathy (TA-TMA) are associated with excessive activation of the alternative complement pathway (AP) and with severe renal, but rarely cerebral, microvascular damage. Here, we compared AP activation and regulation in human glomerular and brain microvascular endothelial cells (GMVECs and BMVECs, respectively) unstimulated or stimulated by the proinflammatory cytokine, tumor necrosis factor (TNF). Compared with GMVECs and under both experimental conditions, BMVECs had increased gene expression of the AP-related genes C3, CFB, and C5 and decreased expression of CFD This was associated with increased expression in BMVECs (relative to GMVECs) of the genes for surface and soluble regulatory molecules (CD46, THBD, CD55, CFI, and CFH) suppressing formation of the AP C3 and C5 convertases. Of note, unlike GMVECs, BMVECs generated extremely low levels of C3a and C5a and displayed decreased activation of the AP (as measured by a lower percentage of Ba generation than GMVECs). Moreover, BMVECs exhibited increased function of CD141, mediating activation of the natural anticoagulant protein C, compared with GMVECs. We also found that the C3a receptor (C3aR) is present on both cell types and that TNF greatly increases C3AR1 expression in GMVECs, but only slightly in BMVECs. Higher AP activation and C3a generation in GMVECs than in BMVECs, coupled with an increase in C3aR production in TNF-stimulated GMVECs, provides a possible explanation for the predominance of renal damage, and the absence of cerebral injury, in individuals with episodes of aHUS and TA-TMA.

Diseases of complement dysregulation-an overview.

Atypical hemolytic uremic syndrome (aHUS), C3 glomerulopathy (C3G), and paroxysmal nocturnal hemoglobinuria (PNH) are prototypical disorders of complement dysregulation. Although complement overactivation is common to all, cell surface alternative pathway dysregulation (aHUS), fluid phase alternative pathway dysregulation (C3G), or terminal pathway dysregulation (PNH) predominates resulting in the very different phenotypes seen in these diseases. The mechanism underlying the dysregulation also varies with predominant acquired autoimmune (C3G), somatic mutations (PNH), or inherited germline mutations (aHUS) predisposing to disease. Eculizumab has revolutionized the treatment of PNH and aHUS although has been less successful in C3G. With the next generation of complement therapeutic in late stage development, these archetypal complement diseases will provide the initial targets.

A haplotype in CFH family genes confers high risk of rare glomerular nephropathies.

Despite distinct renal lesions, a series of rare glomerular nephropathies are reportedly mediated by complement overactivation. Genetic variations in complement genes contribute to disease risk, but the relationship of genotype to phenotype has not been straightforward. Here, we screened 11 complement genes from 91 patients with atypical hemolytic uremic syndrome (aHUS), C3 glomerulopathy (C3G) and membranoproliferative glomerulonephritis type I (MPGN I), and identified the concomitant presence of three missense variations located within the human complement Factor H (CFH) gene cluster. The three variations, rs55807605, rs61737525 and rs57960694, have strong linkage disequilibrium; subsequent haplotype analysis indicated that ATA increased the susceptibility of these renal diseases. In silico analysis, the CFHR3 rs61737525-T risk allele altered the physical and structural properties and generated a reduction in binding affinity of the CFHR3/C3b complex. Surface plasmon resonance (SPR) binding analysis further demonstrated the substitution induced a decrease of two orders of magnitude in C3b-binding properties, with a declined cofactor activity in fluid phase. These data suggest that the haplotype carrying the causative allele behaves as a partial C3 convertase deficiency, predisposing individuals to diverse pathologic lesions underlying complement overactivation. Such genotype-phenotype discrepancies allow better understanding about these nephropathies mediated by genetic complement disorders.

Disease-linked mutations in factor H reveal pivotal role of cofactor activity in self-surface-selective regulation of complement activation.

Spontaneous activation enables the complement system to respond very rapidly to diverse threats. This activation is efficiently suppressed by complement factor H (CFH) on self-surfaces but not on foreign surfaces. The surface selectivity of CFH, a soluble protein containing 20 complement-control protein modules (CCPs 1-20), may be compromised by disease-linked mutations. However, which of the several functions of CFH drives this self-surface selectivity remains unknown. To address this, we expressed human CFH mutants in Pichia pastoris We found that recombinant I62-CFH (protective against age-related macular degeneration) and V62-CFH functioned equivalently, matching or outperforming plasma-derived CFH, whereas R53H-CFH, linked to atypical hemolytic uremic syndrome (aHUS), was defective in C3bBb decay-accelerating activity (DAA) and factor I cofactor activity (CA). The aHUS-linked CCP 19 mutant D1119G-CFH had virtually no CA on (self-like) sheep erythrocytes (ES) but retained DAA. The aHUS-linked CCP 20 mutant S1191L/V1197A-CFH (LA-CFH) had dramatically reduced CA on ES but was less compromised in DAA. D1119G-CFH and LA-CFH both performed poorly at preventing complement-mediated hemolysis of ES PspCN, a CFH-binding Streptococcus pneumoniae protein domain, binds CFH tightly and increases accessibility of CCPs 19 and 20. PspCN did not improve the DAA of any CFH variant on ES Conversely, PspCN boosted the CA, on ES, of I62-CFH, R53H-CFH, and LA-CFH and also enhanced hemolysis protection by I62-CFH and LA-CFH. We conclude that CCPs 19 and 20 are critical for efficient CA on self-surfaces but less important for DAA. Exposing CCPs 19 and 20 with PspCN and thus enhancing CA on self-surfaces may reverse deficiencies of some CFH variants.

Eculizumab in secondary atypical haemolytic uraemic syndrome.

Background: Complement dysregulation occurs in thrombotic microangiopathies (TMAs) other than primary atypical haemolytic uraemic syndrome (aHUS). A few of these patients have been reported previously to be successfully treated with eculizumab. Methods: We identified 29 patients with so-called secondary aHUS who had received eculizumab at 11 Spanish nephrology centres. Primary outcome was TMA resolution, defined by a normalization of platelet count (>150 × 10 9 /L) and haemoglobin, disappearance of all the markers of microangiopathic haemolytic anaemia (MAHA), and improvement of renal function, with a ≥25% reduction of serum creatinine from the onset of eculizumab administration. Results: Twenty-nine patients with secondary aHUS (15 drug-induced, 8 associated with systemic diseases, 2 with postpartum, 2 with cancer-related, 1 associated with acute humoral rejection and 1 with intestinal lymphangiectasia) were included in this study. The reason to initiate eculizumab treatment was worsening of renal function and persistence of TMA despite treatment of the TMA cause and plasmapheresis. All patients showed severe MAHA and renal function impairment (14 requiring dialysis) prior to eculizumab treatment and 11 presented severe extrarenal manifestations. A rapid resolution of the TMA was observed in 20 patients (68%), 15 of them showing a ≥50% serum creatinine reduction at the last follow-up. Comprehensive genetic and molecular studies in 22 patients identified complement pathogenic variants in only 2 patients. With these two exceptions, eculizumab was discontinued, after a median of 8 weeks of treatment, without the occurrence of aHUS relapses. Conclusion: Short treatment with eculizumab can result in a rapid improvement of patients with secondary aHUS in whom TMA has persisted and renal function worsened despite treatment of the TMA-inducing condition.