Atypical hemolytic uremic syndrome (aHUS) usually results from an overactivation of the alternative complement pathway. As large clinical trials are scarce, patient registries can partially fill the knowledge gap on patient characteristics, management, and outcomes. We here describe the baseline clinical and genetic characteristics as well as the management of all Belgian patients enrolled in the Global aHUS Registry at data cut-off. This observational study prospectively and retrospectively collected data (data cut-off: December 26, 2022) from patients of all ages with a clinical diagnosis of aHUS, irrespective of treatment. A total of 121 Belgian patients were registered in the Global aHUS Registry, resulting in a prevalence of 10.4 aHUS patients per million inhabitants, with a higher proportion of females affected (57.9

Scleroderma renal crisis is a severe complication of systemic sclerosis with a poor prognosis. Therefore, identifying precipitating factors is essential. Among known risk factors, only few are reversible. On the contrary, anti-C5 therapy appears effective, at least in some cases. We describe a 59-year-old man with diffuse cutaneous systemic sclerosis who developed life-threatening scleroderma renal crisis following ibuprofen administration. Despite aggressive management, he did not improve. Renal biopsy have displayed features of thrombotic microangiopathy but no complement deposition. We then discuss the pathomechanism of scleroderma renal crisis that could drive eculizumab treatment since some renal biopsies exhibit complement deposits and others do not.

Improvements in COVID-19 treatments, especially for the critically ill, require deeper understanding of the mechanisms driving disease pathology. The complement system is not only a crucial component of innate host defense but can also contribute to tissue injury. Although all complement pathways have been implicated in COVID-19 pathogenesis, the upstream drivers and downstream effects on tissue injury remain poorly defined. We demonstrate that complement activation is primarily mediated by the alternative pathway, and we provide a comprehensive atlas of the complement alterations around the time of respiratory deterioration. Proteomic and single-cell sequencing mapping across cell types and tissues reveals a division of labor between lung epithelial, stromal, and myeloid cells in complement production, in addition to liver-derived factors. We identify IL-6 and STAT1/3 signaling as an upstream driver of complement responses, linking complement dysregulation to approved COVID-19 therapies. Furthermore, an exploratory proteomic study indicates that inhibition of complement C5 decreases epithelial damage and markers of disease severity. Collectively, these results support complement dysregulation as a key druggable feature of COVID-19.

To improve COVID-19 therapy, it is essential to understand the mechanisms driving critical illness. The complement system is an essential part of innate host defense that can also contribute to injury. All complement pathways have been implicated in COVID-19 pathogenesis, however the upstream drivers and downstream consequences on tissue injury remain ill-defined. Here, we demonstrate that complement activation is mediated by the alternative pathway and we provide a comprehensive atlas of the alterations in complement around the time of respiratory deterioration. Proteome and single-cell sequencing mapping across cell types and tissues reveals a division of labor between lung epithelial, stromal and myeloid cells in the production of complement, in addition to liver-derived factors. Upstream, IL-6 drives complement responses, linking complement dysregulation to approved COVID-19 therapies. In an exploratory proteomic study, C5 inhibition improves epithelial damage and markers of disease severity. Collectively, these results identify complement dysregulation as a key druggable feature of COVID-19.### Competing Interest StatementBNL received consulting fees from Sanofi and GSK and holds stock options from Argenx. All remaining authors do not report conflicts of interest.### Funding StatementVIB Tech Watch Fund supported the Olink proteomics (proximity extension analysis). The biomarker studies were funded by the VIB Grand Challenges program (M901BALA -GCP-COVID-19-SARPAC TRIAL and M902BALA-GCP-COVID-19-IL6-IL1 TRIAL). The single-cell CITE-seq profiling of BAL cells was funded through the Chan Zuckerberg Initiative (CZI) Covid atlas project (2020-216717), UGent COVID grant Covid-Track project (BOFCOV; 01C04620), FWO COVID grant Covid-Trace project (G0G4520N), and Ghent University GOA project (01G02418). Partner Therapeutics provided study medication for the SARPAC trial. COV-AID was funded by the Belgian Health Care Knowledge Center. The ZILUCOV trial was funded by UCB. Long-term follow-up of all trial participants was supported by a Ghent University Hospital grant (BOFCOV2020000801). KVD, LH, JD, EDL are beneficiaries of FWO predoctoral fellowships. STTS and SJT are supported by FWO postdoctoral fellowships, and STTS received funding from NWO (452019321). BNL received an European Research Council Advanced Grant (ERC-2017-ADG-789384), several FWO grants and a University of Ghent Methusalem Grant.### Author DeclarationsI confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.YesThe details of the IRB/oversight body that provided approval or exemption for the research described are given below:All studies were approved by the competent authorities and the Ethical Committee of Ghent University Hospital, and all trials were conducted in accordance with Good Clinical Practice guidelines and the Declaration of Helsinki.I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals.YesI understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).YesI have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable.YesAll data produced in the present study are available upon reasonable request to the authors.

IntroductionThrombotic microangiopathy (TMA) is a systemic disorder characterized by either thrombotic or nonthrombotic microvascular lesions, leading to microangiopathic hemolytic anemia, thrombocytopenia, and ischemic organ involvement.1Goodship T.H.J. Cook H.T. Fakhouri F. et al.Atypical hemolytic uremic syndrome and C3 glomerulopathy: conclusions from a "Kidney Disease: Improving Global Outcomes" (KDIGO) Controversies Conference.Kidney Int. 2017; 91: 539-551https://doi.org/10.1016/j.kint.2016.10.005Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar Atypical hemolytic uremic syndrome is characterized by a deficient regulation and therefore an hyperactivation of the alternative pathway of the complement leading to endothelial injury.1Goodship T.H.J. Cook H.T. Fakhouri F. et al.Atypical hemolytic uremic syndrome and C3 glomerulopathy: conclusions from a "Kidney Disease: Improving Global Outcomes" (KDIGO) Controversies Conference.Kidney Int. 2017; 91: 539-551https://doi.org/10.1016/j.kint.2016.10.005Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar,2Noris M. Remuzzi G. Glomerular diseases dependent on complement activation, including atypical hemolytic uremic syndrome, membranoproliferative glomerulonephritis, and C3 glomerulopathy: core curriculum 2015.Am J Kidney Dis. 2015; 66: 359-375https://doi.org/10.1053/j.ajkd.2015.03.040Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar More than 50% of patients with atypical hemolytic uremic syndrome have a proved mutation in the alternative pathway of the complement regulatory genes or other noncomplement related genes.1Goodship T.H.J. Cook H.T. Fakhouri F. et al.Atypical hemolytic uremic syndrome and C3 glomerulopathy: conclusions from a "Kidney Disease: Improving Global Outcomes" (KDIGO) Controversies Conference.Kidney Int. 2017; 91: 539-551https://doi.org/10.1016/j.kint.2016.10.005Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar,2Noris M. Remuzzi G. Glomerular diseases dependent on complement activation, including atypical hemolytic uremic syndrome, membranoproliferative glomerulonephritis, and C3 glomerulopathy: core curriculum 2015.Am J Kidney Dis. 2015; 66: 359-375https://doi.org/10.1053/j.ajkd.2015.03.040Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar Many drugs have been associated with TMA in the literature (Table 11Goodship T.H.J. Cook H.T. Fakhouri F. et al.Atypical hemolytic uremic syndrome and C3 glomerulopathy: conclusions from a "Kidney Disease: Improving Global Outcomes" (KDIGO) Controversies Conference.Kidney Int. 2017; 91: 539-551https://doi.org/10.1016/j.kint.2016.10.005Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar,2Noris M. Remuzzi G. Glomerular diseases dependent on complement activation, including atypical hemolytic uremic syndrome, membranoproliferative glomerulonephritis, and C3 glomerulopathy: core curriculum 2015.Am J Kidney Dis. 2015; 66: 359-375https://doi.org/10.1053/j.ajkd.2015.03.040Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar,S1). The causal relationship is however uncertain for many drugs because this complication is rare and can appear many years after the initiation of the drug and therefore cannot be accessible for randomized trials.3Parisi M. Manni A. Caputo F. et al.A case report of late-onset atypical hemolytic uremic syndrome during interferon beta in multiple sclerosis: open issues in literature review.Brain Behav. 2021; 11: e01930https://doi.org/10.1002/brb3.1930Crossref PubMed Scopus (2) Google Scholar Drug-mediated TMA can be immune mediated or secondary to direct endothelial cell toxicity.4George J.N. Morton J.M. Liles N.W. Nester C.M. After the party's over.N Engl J Med. 2017; 376: 74-80https://doi.org/10.1056/NEJMcps1606750Crossref PubMed Scopus (12) Google Scholar The latter mechanism seems to be implicated in interferon beta-1a (IFN β-1a)–associated TMA.5Jia H. Thelwell C. Dilger P. et al.Endothelial cell functions impaired by interferon in vitro: insights into the molecular mechanism of thrombotic microangiopathy associated with interferon therapy.Thromb Res. 2018; 163: 105-116https://doi.org/10.1016/j.thromres.2018.01.039Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar Interferon-beta1a is an immune-modulating agent widely used as a first-line treatment for relapsing–remitting multiple sclerosis.6Jakimovski D. Kolb C. Ramanathan M. et al.Interferon β for multiple sclerosis.Cold Spring Harb Perspect Med. 2018; 8: a032003https://doi.org/10.1101/cshperspect.a032003Crossref PubMed Scopus (64) Google ScholarTable 1Etiologies of secondary TMA1Goodship T.H.J. Cook H.T. Fakhouri F. et al.Atypical hemolytic uremic syndrome and C3 glomerulopathy: conclusions from a "Kidney Disease: Improving Global Outcomes" (KDIGO) Controversies Conference.Kidney Int. 2017; 91: 539-551https://doi.org/10.1016/j.kint.2016.10.005Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar,2Noris M. Remuzzi G. Glomerular diseases dependent on complement activation, including atypical hemolytic uremic syndrome, membranoproliferative glomerulonephritis, and C3 glomerulopathy: core curriculum 2015.Am J Kidney Dis. 2015; 66: 359-375https://doi.org/10.1053/j.ajkd.2015.03.040Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar,S1InfectiousHIVHepatitis A, CInfluenza H1N1CMVEBVPneumococcus (positive direct Coombs test)COVID-19NeoplasiaParaneoplastic syndrome (antifactor H antibodies)Systemic or autoimmuneSystemic lupus erythematousSclerodermaAntiphospholipid syndromeC3 nephropathyIgA nephropathyMalignant hypertensionDrug inducedMitomycinGemcitabinCalcineurin inhibitorAnti-VEGFQuininTiclopidinInterferon alpha and betaCocaineEstroprogestativeMetabolic diseaseCobalamin C deficiencyPost transplantationStem cell transplantationOrgan transplantationRenal transplantationPregnancyHELLP syndrome/pre-eclampsiaPregnancy relatedCMV, cytomegalovirus; EBV, Epstein-Barr virus; HELLP, hemolysis, elevated liver enzymes, low platelet count;VEGF, vascular endothelial growth factor. Open table in a new tab Case PresentationA 48-year-old male was admitted to the emergency department for focal epileptic seizure. His medical history included a 13-year history of relapsing–remitting multiple sclerosis, treated with IFN β-1a (Avonex, Biogen, Netherlands) 30 μg weekly at diagnosis time and increased to another IFN β-1a (Rebif, Merck, Europe) at the dose of 44 μg, thrice weekly after a few years because of the persistent activity of the disease. He was also treated with levetiracetam 500 mg twice daily for secondary epilepsy. Blood pressure level was normal and physical examination result was unremarkable. Laboratory findings and urine analysis at the admission and 1 month before admission are found in Table 2. Renal biopsy result revealed severe TMA lesions at light and electron microscopy, with negative immunofluorescence staining (Figure 1a-d and Supplementary Figure S1). ADAMTS13 activity was normal, and no Shigatoxin was found in the stool and urine. Complement system analysis revealed an elevated factor B and factor Bb with a normal FBb to Fb ratio. SC5b-9 was also elevated (Supplementary Table S1). Genetic workup did not find a mutation for regulators of the ACP.Table 2Blood and urine analysis at admissionParametersAdmission values1 mo before the admissionNormal valuesBlood analysisHemoglobin (g/dl)7.011.213–18MCV (fl)908980–100Thrombocytes/μl148,000182,000150–440,000Leucocytes/μl16,06080003500–11,000CRP (mg/dl)1.310<5PT (%)/aPTT (s)120/23.7Missing value70–100/21.6–28.7Fibrinogen (mg/dl)486Missing value150–400Urea (mg/dl)631917–48Creatinine (mg/dl)2.941.10.7–1.2K/HCO3 (mmol/l)4/213.7/3.83.5–4.5/23–29LDH (UI/l)918199135–225Haptoglobin (mg/dl)<10Missing value30–200Schistocytes (per 1000 erythrocytes)25/1000Missing value<10/1000Urine analysisProteinuria (g/g de creatinine)5.6<0.3<0.3Albuminuria (mg/g de creatinine)3900<30<30Erythrocytes/μl28Missing value<12Leucocytes/μl<10Missing value<10Pathologic cylindersPresenceMissing valueAbsenceUrea ER (%)42.5Missing value<35Sodium ER (%)2.8Missing value<1aPTT, activated partial thromboplastin time; CRP, C-reactive protein; ER, excretion ratio; HCO3, bicarbonate; K, potassium; LDH, lactate dehydrogenase; MCV, mean corpuscular volume (femtoliters); PT, prothrombin time. Open table in a new tab Because IFN-mediated TMA was highly suspected, its administration was withdrawn and was followed by rapid spontaneous resolution of hemolytic microangiopathic anemia and thrombocytopenia within 2 days. No plasma exchange was required and no eculizumab was administered in the setting of secondary TMA. Renal function continued to decline. After 10 days of admission, the patient developed oliguria with severe hypervolemia requiring initiation of hemodialysis. Supplementary Figure S2 reveals the evolution of renal function and hemolytic microangiopathic anemia parameters. No relapse of TMA was observed during the next 5 months of follow-up. His renal function slowly recovered, and hemodialysis was stopped after 2 months with persistence of a chronic kidney disease stage G3b (estimated glomerular filtration rate 31 ml/min per 1.73 m2 according to Chronic Kidney Disease-Epidemiology Collaboration). The patient experienced 2 other episodes of generalized seizures during the follow-up and died because of a pulmonary septic shock.DiscussionOur patient presented classical biological and histologic TMA. Histologic findings are not specific and therefore cannot help for differential diagnosis.1Goodship T.H.J. Cook H.T. Fakhouri F. et al.Atypical hemolytic uremic syndrome and C3 glomerulopathy: conclusions from a "Kidney Disease: Improving Global Outcomes" (KDIGO) Controversies Conference.Kidney Int. 2017; 91: 539-551https://doi.org/10.1016/j.kint.2016.10.005Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar IFN-mediated TMA is a well-known but rare entity, first described with type I alpha-IFN. IFN β-1a–mediated TMA was first described in 1998,7Ubara Y. Hara S. Takedatu H. et al.Hemolytic uremic syndrome associated with beta-interferon therapy for chronic hepatitis C.Nephron. 1998; 80: 107-108https://doi.org/10.1159/000045147Crossref PubMed Scopus (30) Google Scholar and approximately 30 cases have been reported since then. On the basis of limited case reports and case series in literature, IFN β-1a–mediated TMA occurs after several years of a well-tolerated treatment (mean duration of 11 years), as in our patient.3Parisi M. Manni A. Caputo F. et al.A case report of late-onset atypical hemolytic uremic syndrome during interferon beta in multiple sclerosis: open issues in literature review.Brain Behav. 2021; 11: e01930https://doi.org/10.1002/brb3.1930Crossref PubMed Scopus (2) Google Scholar It is also more frequent in women and occurs mostly in young adults (mean age of 39 years old). There are no guidelines for the treatment of IFN β-1a–mediated TMA. Withdrawal of the drug is key. Plasma exchange and corticosteroids are often used with poor renal outcome.3Parisi M. Manni A. Caputo F. et al.A case report of late-onset atypical hemolytic uremic syndrome during interferon beta in multiple sclerosis: open issues in literature review.Brain Behav. 2021; 11: e01930https://doi.org/10.1002/brb3.1930Crossref PubMed Scopus (2) Google Scholar,8Allinovi M. Cirami C.L. Caroti L. et al.Thrombotic microangiopathy induced by interferon beta in patients with multiple sclerosis: three cases treated with eculizumab.Clin Kidney J. 2017; 10: 625-631https://doi.org/10.1093/ckj/sfw143Crossref PubMed Scopus (16) Google Scholar Approximately 20% of patients will recover with normal renal function, approximately one-third will experience chronic kidney disease, and approximately 40% will have end-stage renal disease.8Allinovi M. Cirami C.L. Caroti L. et al.Thrombotic microangiopathy induced by interferon beta in patients with multiple sclerosis: three cases treated with eculizumab.Clin Kidney J. 2017; 10: 625-631https://doi.org/10.1093/ckj/sfw143Crossref PubMed Scopus (16) Google ScholarKavanagh et al.9Kavanagh D. McGlasson S. Jury A. et al.Type I interferon causes thrombotic microangiopathy by a dose-dependent toxic effect on the microvasculature.Blood. 2016; 128: 2824-2833https://doi.org/10.1182/blood-2016-05-715987Crossref PubMed Scopus (61) Google Scholar investigated the causal relationship between IFN β-1a and TMA. First, in a cohort of patients treated with IFN β-1a for relapsing–remitting multiple sclerosis, there were significantly higher weight-adjusted doses of IFN β-1a in patients with TMA, compared with patients without TMA. Second, among 15 patients with IFN β-1a–induced TMA, none were treated with low doses of IFN β-1a (<50 mcg weekly), 8% were treated with 66 mcg, and 92% with 132 mcg. Finally, they created a transgenic mouse model with mice producing type I interferon either at low levels (IFNlow) or high levels (IFNhigh). In comparison to wild-type mice, they confirmed the dose-dependent relationship on renal microvasculature lesions. They also crossed IFNhigh mice with mice that were null for the type I interferon receptor (IFNAR−/−), confirming the implication of the latter receptor in up-regulation of interferon response genes and histologic lesions. Indeed, IFNhigh × IFNAR−/− mice did not have any microvascular histologic lesion. Jia et al.5Jia H. Thelwell C. Dilger P. et al.Endothelial cell functions impaired by interferon in vitro: insights into the molecular mechanism of thrombotic microangiopathy associated with interferon therapy.Thromb Res. 2018; 163: 105-116https://doi.org/10.1016/j.thromres.2018.01.039Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar revealed that IFN β-1a was associated with endothelial cell dysfunction and lower survival by the inhibition of fibrinolysis and vascular endothelial growth factor-dependent angiogenesis in an in vitro study using human umbilical vein endothelial cells. These findings are also consistent with the association of vasulopathy and TMA in rare interferonopathic diseases, such as Aicardy-Goutière syndrome.9Kavanagh D. McGlasson S. Jury A. et al.Type I interferon causes thrombotic microangiopathy by a dose-dependent toxic effect on the microvasculature.Blood. 2016; 128: 2824-2833https://doi.org/10.1182/blood-2016-05-715987Crossref PubMed Scopus (61) Google Scholar,S1,S2Is there a relationship between IFN β-1a and ACP activation? Parisi et al.3Parisi M. Manni A. Caputo F. et al.A case report of late-onset atypical hemolytic uremic syndrome during interferon beta in multiple sclerosis: open issues in literature review.Brain Behav. 2021; 11: e01930https://doi.org/10.1002/brb3.1930Crossref PubMed Scopus (2) Google Scholar reported 25 cases of TMA in patients treated with IFN. Among these patients, 18 were treated with IFN β-1a and 6 had atypical hemolytic uremic syndrome (defined as the absence of thrombotic thrombocytopenic purpura, hemolytic uremic syndrome, or other secondary TMA except of IFN). Interestingly, mutations for regulators of the ACP were assessed in 5 of these patients. One patient had a heterozygous mutation of the MCP of unknown significance. Another patient had a nonpathogenic heterozygous deletion of CFHR1/R3.8Allinovi M. Cirami C.L. Caroti L. et al.Thrombotic microangiopathy induced by interferon beta in patients with multiple sclerosis: three cases treated with eculizumab.Clin Kidney J. 2017; 10: 625-631https://doi.org/10.1093/ckj/sfw143Crossref PubMed Scopus (16) Google Scholar CFHR1/3 is known to be associated with antifactor H antibodies, but that was not the case in this patient. Genetic workup was negative in 3 patients and missing in 1 case report.3Parisi M. Manni A. Caputo F. et al.A case report of late-onset atypical hemolytic uremic syndrome during interferon beta in multiple sclerosis: open issues in literature review.Brain Behav. 2021; 11: e01930https://doi.org/10.1002/brb3.1930Crossref PubMed Scopus (2) Google Scholar Interestingly, among these 6 patients, 4 were treated with eculizumab, an anticomplement C5 monoclonal antibody. IFN was withdrawn in all patients, and all were treated with plasma exchange (8–18 sessions). One patient was treated with corticosteroid because of worsening of renal function requiring the initiation of hemodialysis. Despite the absence of proven mutation for regulators of the ACP, eculizumab was associated with a significant improvement of renal function, with persistence of chronic kidney disease stages 2 to 4. Hemodialysis was stopped 5 months after the initiation of eculizumab in the most severe case.3Parisi M. Manni A. Caputo F. et al.A case report of late-onset atypical hemolytic uremic syndrome during interferon beta in multiple sclerosis: open issues in literature review.Brain Behav. 2021; 11: e01930https://doi.org/10.1002/brb3.1930Crossref PubMed Scopus (2) Google Scholar,8Allinovi M. Cirami C.L. Caroti L. et al.Thrombotic microangiopathy induced by interferon beta in patients with multiple sclerosis: three cases treated with eculizumab.Clin Kidney J. 2017; 10: 625-631https://doi.org/10.1093/ckj/sfw143Crossref PubMed Scopus (16) Google Scholar These data suggest a potential efficacy of eculizumab but are not a proof for a causal relationship. Our patient did not receive eculizumab despite a potential alternative complement pathway activation (Supplementary Table S1). Indeed, these analyses were performed 4 days after cefuroxime was started for a pyelonephritis, and therefore their interpretation remains uncertain. Moreover, TMA resolved rapidly and spontaneously, and the reimbursement policies of social security in Belgium do not allow prescription of eculizumab in a patient with secondary TMA.Pathophysiological Hypotheses of IFN β-1a–Mediated TMA1.IFN β-1a could act as a trigger in patients with yet undiscovered mutation for regulators of the ACP. Indeed, complement-mediated TMA is usually triggered by a second hit such as infection, pregnancy, or the initiation of a new drug.S3,S4 IFN β-1a may act as a second hit, by its direct toxicity on endothelial cells9Kavanagh D. McGlasson S. Jury A. et al.Type I interferon causes thrombotic microangiopathy by a dose-dependent toxic effect on the microvasculature.Blood. 2016; 128: 2824-2833https://doi.org/10.1182/blood-2016-05-715987Crossref PubMed Scopus (61) Google Scholar and indirect action on fibrinolysis and angiogenesis.5Jia H. Thelwell C. Dilger P. et al.Endothelial cell functions impaired by interferon in vitro: insights into the molecular mechanism of thrombotic microangiopathy associated with interferon therapy.Thromb Res. 2018; 163: 105-116https://doi.org/10.1016/j.thromres.2018.01.039Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar2.IFN β-1a may activate the complement system:•A direct activation of the complement cascade by IFN β-1a has been described,S5 suggesting a potential direct crosstalk between IFN and complement cascade.•An indirect activation of the complement cascade can also be hypothesized. Malignant hypertension may activate complement pathway,S6 but IFN β-1a is not associated with such complication in literature. Antiphospholipid antibodies may also be a potential factor. The latter has been associated with IFN β therapy,S7 is a well-known cause of TMA,1Goodship T.H.J. Cook H.T. Fakhouri F. et al.Atypical hemolytic uremic syndrome and C3 glomerulopathy: conclusions from a "Kidney Disease: Improving Global Outcomes" (KDIGO) Controversies Conference.Kidney Int. 2017; 91: 539-551https://doi.org/10.1016/j.kint.2016.10.005Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar and is known to activate the complement pathway.S8 Our patient had 1 antiphospholipid positive assay at the time IFN β-1a was started. Control result was negative at 12 weeks, and since then, several antiphospholipid assay results were negative.•Complement cascade activation has been described in some drugs, in the setting of secondary TMA (e.g., gemcitabine or cisplatin).S9•Complement system dysregulation seems to be implicated in the pathogenesis of multiple sclerosis.S10 Classical pathway seems to be activated and patients with multiple sclerosis may experience plasma elevation of C3, C4, C4a, C5b-9/MAC, and factor H.S11 Our patient had elevated factor B, factor Bb, and serum C5b-9, suggesting an activation of the alternative pathway of the complement. Plasma C3 level was normal. These data could be associated with multiple sclerosis itself. Indeed, experimental studies have revealed that factor B could be implicated in the pathogenesis of multiple sclerosis.S12 Our patient's neurologic assessment revealed no active lesion at magnetic resonance imaging and no worsening of neurologic status that were consistent with a nonactive disease. Complement activation could also be associated with the episode of pyelonephritis that our patient presented. Finally, as discussed previously, the complement activation has been associated with IFN β-1a treatment in the setting of TMA (summary illustrated in Supplementary Figures S2, S3, and S4).Figure 2 summarizes the pathophysiological hypotheses of IFN β-1a–mediated TMA.Figure 2Pathophysiological hypotheses of IFN β-1a–mediated TMA. Blue lines represent data from literature whereas green arrows represent pathophysiological hypotheses. The red arrow represents the absence of data revealing a link between type I IFN and hypertension in literature. IFN, interferon; TMA, thrombotic microangiopathy.View Large Image Figure ViewerDownload Hi-res image Download (PPT)ConclusionPrescribers of IFN β-1a should be aware of IFN β-1a–mediated TMA. Weight-adjusted doses should be evaluated on a regular basis and adjusted according to the disease activity because this complication is dose dependent. Treatment mainly consists in withdrawal of IFN, corticosteroids, and plasma exchange with poor renal outcome. Eculizumab seems to be promising, because it has been associated with a better renal prognosis in case series even in the absence of a demonstrated abnormality in the regulatory factors of the ACP. These data may suggest that either IFN β-1a activates the complement cascade or that TMA occurs as a second hit in a patient with atypical hemolytic uremic syndrome and yet undiscovered mutations for regulators of the ACP. These data need to be confirmed by further studies with a higher level of evidence.DisclosureP.S. reports personal fees from Sanofi Genzyme, outside the submitted work. All the other authors declared no competing interests.Patient ConsentThe patient's next of kin provided consent to publish this case study. IntroductionThrombotic microangiopathy (TMA) is a systemic disorder characterized by either thrombotic or nonthrombotic microvascular lesions, leading to microangiopathic hemolytic anemia, thrombocytopenia, and ischemic organ involvement.1Goodship T.H.J. Cook H.T. Fakhouri F. et al.Atypical hemolytic uremic syndrome and C3 glomerulopathy: conclusions from a "Kidney Disease: Improving Global Outcomes" (KDIGO) Controversies Conference.Kidney Int. 2017; 91: 539-551https://doi.org/10.1016/j.kint.2016.10.005Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar Atypical hemolytic uremic syndrome is characterized by a deficient regulation and therefore an hyperactivation of the alternative pathway of the complement leading to endothelial injury.1Goodship T.H.J. Cook H.T. Fakhouri F. et al.Atypical hemolytic uremic syndrome and C3 glomerulopathy: conclusions from a "Kidney Disease: Improving Global Outcomes" (KDIGO) Controversies Conference.Kidney Int. 2017; 91: 539-551https://doi.org/10.1016/j.kint.2016.10.005Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar,2Noris M. Remuzzi G. Glomerular diseases dependent on complement activation, including atypical hemolytic uremic syndrome, membranoproliferative glomerulonephritis, and C3 glomerulopathy: core curriculum 2015.Am J Kidney Dis. 2015; 66: 359-375https://doi.org/10.1053/j.ajkd.2015.03.040Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar More than 50% of patients with atypical hemolytic uremic syndrome have a proved mutation in the alternative pathway of the complement regulatory genes or other noncomplement related genes.1Goodship T.H.J. Cook H.T. Fakhouri F. et al.Atypical hemolytic uremic syndrome and C3 glomerulopathy: conclusions from a "Kidney Disease: Improving Global Outcomes" (KDIGO) Controversies Conference.Kidney Int. 2017; 91: 539-551https://doi.org/10.1016/j.kint.2016.10.005Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar,2Noris M. Remuzzi G. Glomerular diseases dependent on complement activation, including atypical hemolytic uremic syndrome, membranoproliferative glomerulonephritis, and C3 glomerulopathy: core curriculum 2015.Am J Kidney Dis. 2015; 66: 359-375https://doi.org/10.1053/j.ajkd.2015.03.040Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar Many drugs have been associated with TMA in the literature (Table 11Goodship T.H.J. Cook H.T. Fakhouri F. et al.Atypical hemolytic uremic syndrome and C3 glomerulopathy: conclusions from a "Kidney Disease: Improving Global Outcomes" (KDIGO) Controversies Conference.Kidney Int. 2017; 91: 539-551https://doi.org/10.1016/j.kint.2016.10.005Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar,2Noris M. Remuzzi G. Glomerular diseases dependent on complement activation, including atypical hemolytic uremic syndrome, membranoproliferative glomerulonephritis, and C3 glomerulopathy: core curriculum 2015.Am J Kidney Dis. 2015; 66: 359-375https://doi.org/10.1053/j.ajkd.2015.03.040Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar,S1). The causal relationship is however uncertain for many drugs because this complication is rare and can appear many years after the initiation of the drug and therefore cannot be accessible for randomized trials.3Parisi M. Manni A. Caputo F. et al.A case report of late-onset atypical hemolytic uremic syndrome during interferon beta in multiple sclerosis: open issues in literature review.Brain Behav. 2021; 11: e01930https://doi.org/10.1002/brb3.1930Crossref PubMed Scopus (2) Google Scholar Drug-mediated TMA can be immune mediated or secondary to direct endothelial cell toxicity.4George J.N. Morton J.M. Liles N.W. Nester C.M. After the party's over.N Engl J Med. 2017; 376: 74-80https://doi.org/10.1056/NEJMcps1606750Crossref PubMed Scopus (12) Google Scholar The latter mechanism seems to be implicated in interferon beta-1a (IFN β-1a)–associated TMA.5Jia H. Thelwell C. Dilger P. et al.Endothelial cell functions impaired by interferon in vitro: insights into the molecular mechanism of thrombotic microangiopathy associated with interferon therapy.Thromb Res. 2018; 163: 105-116https://doi.org/10.1016/j.thromres.2018.01.039Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar Interferon-beta1a is an immune-modulating agent widely used as a first-line treatment for relapsing–remitting multiple sclerosis.6Jakimovski D. Kolb C. Ramanathan M. et al.Interferon β for multiple sclerosis.Cold Spring Harb Perspect Med. 2018; 8: a032003https://doi.org/10.1101/cshperspect.a032003Crossref PubMed Scopus (64) Google ScholarTable 1Etiologies of secondary TMA1Goodship T.H.J. Cook H.T. Fakhouri F. et al.Atypical hemolytic uremic syndrome and C3 glomerulopathy: conclusions from a "Kidney Disease: Improving Global Outcomes" (KDIGO) Controversies Conference.Kidney Int. 2017; 91: 539-551https://doi.org/10.1016/j.kint.2016.10.005Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar,2Noris M. Remuzzi G. Glomerular diseases dependent on complement activation, including atypical hemolytic uremic syndrome, membranoproliferative glomerulonephritis, and C3 glomerulopathy: core curriculum 2015.Am J Kidney Dis. 2015; 66: 359-375https://doi.org/10.1053/j.ajkd.2015.03.040Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar,S1InfectiousHIVHepatitis A, CInfluenza H1N1CMVEBVPneumococcus (positive direct Coombs test)COVID-19NeoplasiaParaneoplastic syndrome (antifactor H antibodies)Systemic or autoimmuneSystemic lupus erythematousSclerodermaAntiphospholipid syndromeC3 nephropathyIgA nephropathyMalignant hypertensionDrug inducedMitomycinGemcitabinCalcineurin inhibitorAnti-VEGFQuininTiclopidinInterferon alpha and betaCocaineEstroprogestativeMetabolic diseaseCobalamin C deficiencyPost transplantationStem cell transplantationOrgan transplantationRenal transplantationPregnancyHELLP syndrome/pre-eclampsiaPregnancy relatedCMV, cytomegalovirus; EBV, Epstein-Barr virus; HELLP, hemolysis, elevated liver enzymes, low platelet count;VEGF, vascular endothelial growth factor. 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IntroductionThrombotic microangiopathy (TMA) is a rare systemic microvascular disorder characterized by microangiopathic hemolytic anemia, thrombocytopenia, and ischemic damage in different organs. Beta-interferon (β-IFN), an immune-modulating agent widely used as a first line treatment for relapsing–remitting multiple sclerosis (RRMS), has been associated with TMA in several reports but the underlying mechanisms are still controversial. Treatment is mainly based on plasma exchanges, corticosteroids but the renal prognosis is poor.MethodsWe report the case of a 48-year-old male patient, with a 13-years history of RRMS, treated weekly with 132 µg of a type I Beta-Interferon (βI-IFN) and admitted for focal seizure. Initial investigations revealed classical laboratory features of TMA and a severe acute kidney injury requiring renal replacement therapy. Renal biopsy was performed and showed severe TMA lesions. Thrombotic thrombocytopenic purpura and typical hemolytic uremic syndrome were ruled out. Laboratory findings interestingly suggested a possible involvement of the alternative pathway of the complement (APC) with CFB: 32,5 mg/dl (normal value: 11-22 mg/dl), FBb: 0,258 mg/dl (normal value <0,153 mg/dl) and SC5b-9: 822 ng/ml (normal value <314 ng/ml). Genetic workup did not find a mutation for regulators of the APC, and the search for anti-factor H antibodies was negative. The withdrawal of βI-IFN resulted in the rapid resolution of hemolytic microangiopathic anemia and thrombocytopenia, but with delayed improvement of the renal function. No plasma exchange was required and no eculizumab was administered because of the absence of reimbursement procedure available in our country in case of secondary TMA.ResultsTMA is a rare complication of βI-IFN treatment. Recent experimental data suggest a causal relationship. Kavanagh et al. showed that βI-IFN was responsible for a direct dose-dependent TMA in a rodent model, and that TMA was absent in type I-IFN receptor (IFNAR) knock-out mice. Jia et al. showed that βI-IFN could interfere with endothelial cell by inhibiting fibrinolysis and VEGF-dependent angiogenesis. Furthermore, eculizumab, an anti-C5 antibody blocking complement terminal activation, has been reported to significantly improve renal prognosis in case series even in the absence of mutation of the APC. These data suggest that 1) TMA may occur as a second hit in patients with yet undiscovered mutations of the APC; or 2) IFN could activate complement cascade in patients without mutations of the APC. Indeed, some data suggest a direct activation of the complement cascade by type-I IFN, but an indirect link can also be hypothesized. For example, antiphospholipid antibodies have been associated with βI-IFN treatment and are known to activate complement pathway and to be associated with TMA. Figure 1. summarizes the pathophysiological hypotheses of βI-IFN mediated TMA. Blue lines represent data from the literature, whereas green arrows represent pathophysiological hypotheses. The red arrow represents lacking data up to now.ConclusionsβI-IFN mediated TMA is a rare but well-known complication. βI-IFN has a direct toxic effect on endothelial cells and can also act as a trigger in the setting of APC regulation deficiency. Eculizumab seems to be effective, even in the absence of mutations for regulators of the APC suggesting a potential activation of the complement cascade by IFN.No conflict of interest IntroductionThrombotic microangiopathy (TMA) is a rare systemic microvascular disorder characterized by microangiopathic hemolytic anemia, thrombocytopenia, and ischemic damage in different organs. Beta-interferon (β-IFN), an immune-modulating agent widely used as a first line treatment for relapsing–remitting multiple sclerosis (RRMS), has been associated with TMA in several reports but the underlying mechanisms are still controversial. Treatment is mainly based on plasma exchanges, corticosteroids but the renal prognosis is poor.

The complement system is an essential part of our innate immune system. Three enzymatic activation pathways are described, all converging into a common terminal pathway which causes lysis of the target cell. Late complement deficiencies (LCDs) are typically diagnosed in children or adolescents with invasive meningococcal disease (IMD). However, IMD can also be a first manifestation in adulthood and should prompt for the evaluation of the LCD. We report the case of a young adult with IMD who was found to have a LCD, caused by a compound heterozygous mutation in C6. His vaccination status was optimized and prophylactic antibiotic treatment was initiated. By means of this case, we would like to raise awareness of underlying LCD in (young) adults presenting with IMD by N. meningitidis. Screening for complement deficiencies after IMD, followed by genetic testing, can be lifesaving and allows for genetic counselling. In addition, we discuss the diagnosis and treatment of LCD.

In rare instances, pediatric SARS-CoV-2 infection results in a novel immunodysregulation syndrome termed multisystem inflammatory syndrome in children (MIS-C). We compared MIS-C immunopathology with severe COVID-19 in adults. MIS-C does not result in pneumocyte damage but is associated with vascular endotheliitis and gastrointestinal epithelial injury. In MIS-C, the cytokine release syndrome is characterized by IFNγ and not type I interferon. Persistence of patrolling monocytes differentiates MIS-C from severe COVID-19, which is dominated by HLA-DRlo classical monocytes. IFNγ levels correlate with granzyme B production in CD16+ NK cells and TIM3 expression on CD38+/HLA-DR+ T cells. Single-cell TCR profiling reveals a skewed TCRβ repertoire enriched for TRBV11-2 and a superantigenic signature in TIM3+/CD38+/HLA-DR+ T cells. Using NicheNet, we confirm IFNγ as a central cytokine in the communication between TIM3+/CD38+/HLA-DR+ T cells, CD16+ NK cells, and patrolling monocytes. Normalization of IFNγ, loss of TIM3, quiescence of CD16+ NK cells, and contraction of patrolling monocytes upon clinical resolution highlight their potential role in MIS-C immunopathogenesis.

Background: Hemolytic uremic syndrome (HUS) is rare in neonates. It is probably an under-recognized condition in the early postnatal period as it presents similarly to the most common perinatal asphyxia and to differentiate the two conditions is challenging. We describe the clinical presentation of a potential new subtype of neonatal HUS triggered by hypoxic-ischemic event. Our patient was successfully treated by a single dose of Eculizumab as early as at 9 days of life. Case Report: A 35-weeks infant was born with low hemoglobin and subsequently developed respiratory distress, hypotension, and acidosis. Blood transfusion was administered, acidosis corrected, neurological examination remained reassuring. Few hours later he developed renal failure, macroscopic hematuria, hemobilia, thrombocytopenia and coagulopathy refractory to platelet and fresh frozen plasma transfusions. No infection was found. Haptoglobin was non-measurable, and schistocytes present, complement factors C3, C4 and B were low, FBb increased. HUS was suspected. A single dose of Eculizumab™ was administered on day 9 of life. No genetic mutation of atypical HUS was found. He was discharged with improving renal function and developing cholestasis. Conclusion: In neonates with hemolytic anemia, thrombocytopenia, hematuria and renal failure, HUS should be suspected. In neonatal HUS Eculizumab should be considered as first-line therapy and discontinuation can be considered if no genetic mutation is found and clinical condition improves. In very young patients, cholestasis could appear as potential side effect of Eculizumab™.

The importance of alternative pathway (AP) regulation by complement factor I (FI) is illustrated by the diverse clinical phenotype associated with complete FI deficiency in humans. Patients experience recurrent invasive infections with encapsulated bacteria, but they are also at risk for developing noninfectious diseases such as chronic inflammation (Fig 1, A).1Merle N.S. Noe R. Halbwachs-Mecarelli L. Fremeaux-Bacchi V. Roumenina L.T. Complement system part II: role in immunity.Front Immunol. 2015; 6: 257Crossref PubMed Scopus (442) Google Scholar So far, only 49 patients with complete FI deficiency, a rare autosomal recessive immunodeficiency, have been reported.2El Sissy C. Rosain J. Vieira-Martins P. Bordereau P. Gruber A. Devriese M. et al.Clinical and genetic spectrum of a large cohort with total and sub-total complement deficiencies.Front Immunol. 2019; 10: 1936Crossref PubMed Scopus (13) Google Scholar,3Shields A.M. Pagnamenta A.T. Pollard A.J. OxClinWGS Taylor J.C. Allroggen H. et al.Classical and non-classical presentations of complement factor I deficiency: two contrasting cases diagnosed via genetic and genomic methods.Front Immunol. 2019; 10: 1150Crossref PubMed Scopus (4) Google Scholar Individuals carrying a heterozygous loss-of-function allele, partial or incomplete FI deficiency, are known to have a predisposition toward atypical hemolytic uremic syndrome, although additional mutations in other regulatory complement proteins are often found.4Kavanagh D. Richards A. Noris M. Hauhart R. Liszewski K.M. Karpman D. et al.Characterization of mutations in complement factor I (CFI) associated with hemolytic uremic syndrome.Mol Immunol. 2008; 45: 95-105Crossref PubMed Scopus (102) Google Scholar,5Bienaime F. Dragon-Durey M.A. Regnier C.H. Nilsson S.C. Kwan W.H. Blouin J. et al.Mutations in components of complement influence the outcome of factor I-associated atypical hemolytic uremic syndrome.Kidney Int. 2010; 77: 339-349Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar Interestingly, similar to in mouse studies, renal disease is not reported in complete FI deficiency.6Rose K.L. Paixao-Cavalcante D. Fish J. Manderson A.P. Malik T.H. Bygrave A.E. et al.Factor I is required for the development of membranoproliferative glomerulonephritis in factor H-deficient mice.J Clin Invest. 2008; 118: 608-618PubMed Google Scholar Patients with complete FI deficiency have low functional C3 and hence are unable to aggress the endothelium. Two different types of CFI gene mutations are reported. Type I CFI mutations lead to completely absent or decreased FI protein expression, resulting in a quantitative FI defect.4Kavanagh D. Richards A. Noris M. Hauhart R. Liszewski K.M. Karpman D. et al.Characterization of mutations in complement factor I (CFI) associated with hemolytic uremic syndrome.Mol Immunol. 2008; 45: 95-105Crossref PubMed Scopus (102) Google Scholar In contrast, type II CFI mutations result in normal FI protein expression but impaired FI function. Regardless of the type of mutation, CFI deficiency will be either partial (residual FI function) in heterozygous carriers or complete (total absence of FI function) in homozygous or compound heterozygous carriers. Currently, complete FI deficiency is underdiagnosed on account of a lack of sensitive and specific diagnostic markers, a broad reference interval for FI serum level, and the fact that most CFI mutations produce variable amounts of FI over time.4Kavanagh D. Richards A. Noris M. Hauhart R. Liszewski K.M. Karpman D. et al.Characterization of mutations in complement factor I (CFI) associated with hemolytic uremic syndrome.Mol Immunol. 2008; 45: 95-105Crossref PubMed Scopus (102) Google Scholar The use of laboratory hallmarks based on the specific dysregulation of the complement cascade can overcome these diagnostic challenges (Fig 1, B). The proteolytic FI is a plasma glycoprotein that inhibits the alternative and classic complement pathways by inactivating C3b and C4b in the presence of cofactors such as membrane cofactor protein (MCP [CD46]), complement receptor type 1 (CR1 [CD35]), C4 binding protein (C4BP), and complement factor H (FH). Differences in binding subsites of C3b explain why cofactors such as FH produce only iC3b fragments, whereas in the presence of CR1, FI is able to further degrade iC3b into the final fragments C3c and C3d (Fig 1, B).7Xue X. Wu J. Ricklin D. Forneris F. Di Crescenzio P. Schmidt C.Q. et al.Regulator-dependent mechanisms of C3b processing by factor I allow differentiation of immune responses.Nat Struct Mol Biol. 2017; 24: 643-651Crossref PubMed Scopus (54) Google Scholar We performed a clinical, laboratory, and genetic evaluation in a cohort of 7 patients (patients A-G) with complete FI deficiency (Tables I8Tortajada A. Pinto S. Martínez-Ara J. López-Trascasa M. Sánchez-Corral P. et al.Complement factor H variants I890 and L1007 while commonly associated with atypical hemolytic uremic syndrome are polymorphisms with no functional significance.Kidney Int. 2012; 81: 56-63Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar,9Haerynck F. Stordeur P. Vandewalle J. Van Coster R. Bordon V. De Baets F. et al.Complete factor I deficiency due to dysfunctional factor I with recurrent aseptic meningo-encephalitis.J Clin Immunol. 2013; 33: 1293-1301Crossref PubMed Scopus (10) Google Scholar and II) (see the Case Descriptions section in Online Repository materials at jacionline.org). The majority of the patients presented with invasive bacterial infections, including bacterial (Streptococcus pneumoniae, Neisseria meningitidis, and Streptococcus pyogenes) meningitis (patients A-C) and bacteremia with S pneumoniae (patient D). Patient E was diagnosed with a cutaneous IgA vasculitis that was confirmed by biopsy, and she recently presented with cerebral vasculitis after acute onset of visual disturbances. She also experienced invasive infections (S pyogenes bacteremia, N meningitidis). Patient F experienced recurrent leukocytoclastic vasculitis of the lower extremities (Fig 1, A) and presented with diplopia and ataxia. She was diagnosed with an aseptic brainstem (Bickerstaff) encephalitis that was treated with corticosteroids and plasmapheresis. Interestingly, our group had already reported a similar case of complete FI deficiency with leukocytoclastic vasculitis and recurrent aseptic meningoencephalitis (patient G).9Haerynck F. Stordeur P. Vandewalle J. Van Coster R. Bordon V. De Baets F. et al.Complete factor I deficiency due to dysfunctional factor I with recurrent aseptic meningo-encephalitis.J Clin Immunol. 2013; 33: 1293-1301Crossref PubMed Scopus (10) Google ScholarTable IDemographic, genetic, and clinical data on the study patients with complete FI deficiencyCharacteristicPatient APatient BPatient CPatient DPatient EPatient FPatient GSexFemaleFemaleMaleMaleFemaleFemaleFemaleAge of onset3 mo7 y3 y12 y11 y13 y16 yCFI mutationHomozygous.c.257G>A p.(C86Y)Compound heterozygous.c.355G>T p.(G119∗)Exon deletion 2-13Homozygous.c.257G>A p.(C86Y)Compound heterozygous.c.1367G>T p.(W456L)Exon deletion 2-13Homozygous.c.1015C>T p.(R339∗)Compound heterozygous.c.1367G>T p.(W456L)c.772G>A p.(A258T)Compound heterozygous.c.1019T>C p.(I322L)c.1571A>C p.(D524V)Tortajada et al8Tortajada A. Pinto S. Martínez-Ara J. López-Trascasa M. Sánchez-Corral P. et al.Complement factor H variants I890 and L1007 while commonly associated with atypical hemolytic uremic syndrome are polymorphisms with no functional significance.Kidney Int. 2012; 81: 56-63Abstract Full Text Full Text PDF PubMed Scopus (30) Google ScholarNewTortajada et al8Tortajada A. Pinto S. Martínez-Ara J. López-Trascasa M. Sánchez-Corral P. et al.Complement factor H variants I890 and L1007 while commonly associated with atypical hemolytic uremic syndrome are polymorphisms with no functional significance.Kidney Int. 2012; 81: 56-63Abstract Full Text Full Text PDF PubMed Scopus (30) Google ScholarBienaime et al5Bienaime F. Dragon-Durey M.A. Regnier C.H. Nilsson S.C. Kwan W.H. Blouin J. et al.Mutations in components of complement influence the outcome of factor I-associated atypical hemolytic uremic syndrome.Kidney Int. 2010; 77: 339-349Abstract Full Text Full Text PDF PubMed Scopus (127) Google ScholarNewBienaime et al5Bienaime F. Dragon-Durey M.A. Regnier C.H. Nilsson S.C. Kwan W.H. Blouin J. et al.Mutations in components of complement influence the outcome of factor I-associated atypical hemolytic uremic syndrome.Kidney Int. 2010; 77: 339-349Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar and Kavanagh et al4Kavanagh D. Richards A. Noris M. Hauhart R. Liszewski K.M. Karpman D. et al.Characterization of mutations in complement factor I (CFI) associated with hemolytic uremic syndrome.Mol Immunol. 2008; 45: 95-105Crossref PubMed Scopus (102) Google ScholarHaerynck et al9Haerynck F. Stordeur P. Vandewalle J. Van Coster R. Bordon V. De Baets F. et al.Complete factor I deficiency due to dysfunctional factor I with recurrent aseptic meningo-encephalitis.J Clin Immunol. 2013; 33: 1293-1301Crossref PubMed Scopus (10) Google Scholar Fremeaux-Bacchi et al1Merle N.S. Noe R. Halbwachs-Mecarelli L. Fremeaux-Bacchi V. Roumenina L.T. Complement system part II: role in immunity.Front Immunol. 2015; 6: 257Crossref PubMed Scopus (442) Google ScholarClinical featuresInfectiousInfectiousInfectiousInfectiousInfectious/AutoimmuneAutoimmuneAutoimmuneS pneumoniae meningitisN meningitidis meningitisS pneumoniae, S pyogenes meningitisS pneumoniae otitis and bacteremiaVasculitis (cutaneous, cerebral), S pyogenes bacteremia, N meningitidis meningitisBickerstaff encephalitis, leukocytoclastic vasculitisAseptic meningoencephalitis, leukocytoclastic vasculitis Open table in a new tab Table IIOverview of complement system analysis of the study patients with complete FI deficiencyPathwayPatient APatient BPatient CPatient DPatient EPatient FPatient GReference rangeClassic pathwayCH50 (U/mL)63<13∗New reference values with different standard used in calibration. Boldface indicates lowered values. Italics indicates increased values.18<13∗New reference values with different standard used in calibration. Boldface indicates lowered values. Italics indicates increased values.463223-6341-94∗New reference values with different standard used in calibration. Boldface indicates lowered values. Italics indicates increased values.C4 (g/L)0.250.30.220.180.120.200.190.1-0.4C3 (mg/dL)1923422522425772-156APAP50 (%)000000030-113FB (mg/dL)12245∗New reference values with different standard used in calibration. Boldface indicates lowered values. Italics indicates increased values.21∗New reference values with different standard used in calibration. Boldface indicates lowered values. Italics indicates increased values.11-228-22∗New reference values with different standard used in calibration. Boldface indicates lowered values. Italics indicates increased values.FH (mg/dL)1624301820424637-73Anti-FH (a.u.)49116581484>100000103∗New reference values with different standard used in calibration. Boldface indicates lowered values. Italics indicates increased values.<150∗New reference values with different standard used in calibration. Boldface indicates lowered values. Italics indicates increased values.0-1000-150∗New reference values with different standard used in calibration. Boldface indicates lowered values. Italics indicates increased values.FI (mg/dL)<1.51.6<1.5<1.53.1<1.54.44.0-10.7Terminal pathwaySC5b-9 (ng/mL)104015591147441143510601281<314Degradation productsFactor Bb (mg/dL)0.5440.2620.790.540.740.890.515<0.15Factor Bb/FB ratio21.941.7344.2713.1714.754.2751.5<1.07C3d (mg/dL)<0.4<0.4<0.4<0.4<0.4<0.4<0.4<1.2∗ New reference values with different standard used in calibration. Boldface indicates lowered values. Italics indicates increased values. Open table in a new tab The extended laboratory evaluation of the complement system in all patients is summarized in Table II. In this cohort, all patients (A-G) presented with absent activity in AP complement activity (AP50), and most of them (patients A-E) also had a decreased level classic pathway complement activity (CH50). An analysis of complement pathway components revealed normal C4 and decreased C3 serum concentrations with elevated levels of the soluble membrane attack complex (sC5b-9). This suggests an overactivation of the AP, resulting in a consumptive C3 deficiency. Other laboratory signatures of AP overactivation include overconsumption of factor B (FB), which is cleaved by factor D into the catalytic subunit Bb to form the alternative C3 convertase (C3bBb). Indeed, all patients showed reduced levels of FB and significantly increased Bb concentrations and Bb/FB ratio compatible with overconsumption of FB. Laboratory hallmarks of AP dysregulation require further examination of the complement regulatory proteins, including the soluble inhibitory regulators FH and FI. Serum concentration of FH was significantly reduced in most of our patients (patients A-E). Autoantibodies against FH were found in 2 patients (C and E) and were associated with a FH-related protein 1 (CFHR1) gene deficiency.5Bienaime F. Dragon-Durey M.A. Regnier C.H. Nilsson S.C. Kwan W.H. Blouin J. et al.Mutations in components of complement influence the outcome of factor I-associated atypical hemolytic uremic syndrome.Kidney Int. 2010; 77: 339-349Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar Secondary reduced serum FH concentration has been reported in FI-deficient individuals.10Naked G.M. Florido M.P. Ferreira de Paula P. Vinet A.M. Inostroza J.S. Isaac L. Deficiency of human complement factor I associated with lowered factor H.Clin Immunol. 2000; 96: 162-167Crossref PubMed Scopus (34) Google Scholar All but 1 of the 7 patients (patient G) had significantly decreased values of serum FI. Evaluation of C3b degradation products such as C3d can reveal an underlying FI defect. We measured C3d concentration in patients with CFI mutations or defects in other regulatory complement factors, including FH, CFHR1-R3, and CD46 (Fig 1, C). The group, referred to as the "partial FI deficiency" group, includes patients carrying a heterozygous mutation of the CFI gene alone (n = 2) or together with a CFHR1-R3 deletion (n = 4). The "other regulatory defects" group (n = 24) includes patients carrying a mutation of the following genes: CFH, CFHR1-R3 deletion, C3, and CD46. Plasma C3d levels were undetectable (<0.4 mg/dL) only in patients with a complete FI deficiency (n = 7). Targeted sequencing of the CFI gene confirmed homozygous (A, C, and E) or compound heterozygous (B, D, and G) mutations in the CFI gene (Table II). Nonsense or frameshift mutations and large exon deletions result in mRNA decay or nonfunctional truncated proteins compatible with an absent or decreased protein expression. However, the significance of missense variants is mainly assessed with in silico pathogenicity prediction tools. In these cases, insight derived from the underlying structural mechanisms can provide an understanding of the effect of disease-associated CFI variants. Mapping of the mutated amino acid residues in the quaternary structure of FI in complex with FH and C3b was performed to predict the pathogenic effects of these missense variants (Fig 1, D). Two missense variants (p.I322L and p.W456L) are located at the interface of heavy and light chain and are predicted to destabilize protein folding. Variant p.D524V is located in the serine protease domain (SP) of the FI light chain and can affect its catalytic function. The p.C86Y and p.A258T are located in the heavy chain and are expected to destabilize the protein structure and cause a protein defect. In conclusion, complete complement FI deficiency is a rare immunodeficiency exhibiting a diverse clinical phenotype impeding early diagnosis. In this cohort, we have shown that a laboratory signature of AP overactivation in combination with undetectable plasma C3d levels is associated with a complete FI defect in patients with homozygous or compound heterozygous mutations in the CFI gene. We thank the patients and their families for consenting to this research. Since early childhood, a 9-year-old Moroccan girl from consanguineous parents experienced recurrent respiratory tract infections necessitating intravenous antibiotic treatment. S pneumoniae was once isolated from blood cultures. At 3 months of age she was admitted to the intensive care unit because of acute fever (40°C), vomiting, and lethargy. Magnetic resonance imaging (MRI) of the brain showed a bilateral frontotemporal subdural effusion (Fig 1, A). S pneumoniae was isolated from her blood and cerebrospinal fluid (CSF). She recovered after intravenous ampicillin treatment. There is no family history of recurrent infections or autoimmune diseases. Laboratory work-up showed normal B- and T-cell immunophenotyping, normal concentrations of total serum IgG and IgG subclasses and adequate specific polysaccharide antibody responses against S pneumoniae. A 7-year-old girl was admitted to the intensive care unit because of hypovolemic shock with fever and petechial rash. N meningitidis was isolated from blood and CSF cultures, and she was successfully treated with intravenous cefotaxime. During her first 3 years of life, she experienced recurrent upper respiratory tract infections; intravenous immunoglobulin substitution treatment was initiated on the basis of an underlying IgG3 subclass deficiency. Her father had no history of infections. Her mother did experience recurrent throat infections but had normal immunologic work-up results. A 6-year-old boy from consanguineous Moroccan parents presented with recurrent meningitis caused by S pneumoniae at 3 years of age and S pyogenes at 4 years of age. Moreover, he experienced recurrent ear infections necessitating antibiotics and 1 episode of bacterial pneumonia. During early childhood, a white male experienced recurrent upper and lower respiratory tract infections (>6 episodes per year) requiring antibiotic treatment. He was admitted at 6 years of age because of pneumococcal septicemia that was successfully treated with intravenous ampicillin. At 12 years of age, he presented with impaired consciousness, acute fever (40°C), and vomiting. CSF analysis showed a highly elevated white blood cell count (8340/μL, 66% neutrophils), a normal glucose concentration (63 mg/dL), and an increased protein concentration (153 mg/dL). Cultures of blood and CSF remained negative. The patient was treated with intravenous ceftriaxone. Further laboratory work-up showed no hematologic abnormalities, normal total serum levels of IgG and IgG subclasses, and a normal antibody response against pneumococcal polysaccharides after vaccination. At 20 years of age, he presented at the emergency department because of a headache, high fever, and lethargy. The result of quantitative PCR of CSF for varicella zoster virus was positive. He recovered with acyclovir treatment. An 18-year-old Moroccan girl had experienced petechiae on the lower extremities since the age of 11 and was diagnosed with IgA vasculitis on the basis of a skin biopsy. Three months later she was hospitalized because of a skin abscess on her right upper arm that was complicated by a group A β-hemolytic Streptococcus bacteremia and a septic arthritis of the left knee. She was successfully treated with intravenous flucloxacillin. Recently, she was admitted for a meningococcal A meningitis and septicemia with a complete clinical recovery. In addition, MRI of the brain because of acute visual disturbances revealed several hyperintense lesions on fluid-attenuated inversion recovery images at the infratentorial and supratentorial region that were suspected as being cerebral vasculitis. Since the age of 7 years, this girl has presented with recurrent cutaneous leukocytoclastic vasculitis on the lower extremities (Fig 1, C). At age 13, she presented at the emergency department with fever (38.4°C), vomiting, and diplopia. Clinical neurologic examination on admission revealed reduced knee jerk reflex and ophthalmologic evaluation showed diplopia as well as horizontal and vertical nystagmus during eye movements. CSF analysis showed an elevated white blood cell count (500/μL, 99% neutrophils), normal glucose concentration (75 mg/dL), and an increased protein concentration (180 mg/dL). Multiplex PCR analysis of her CSF did not detect any pathogens. MRI neuroimaging revealed increased signal intensity in the basal ganglia, the thalamus, and the brainstem to the level of the upper and middle cerebellar peduncles. During treatment with intravenous cefotaxime (1g every 6 hours) and acyclovir (600 mg every 8 hours), she deteriorated after 10 days, with loss of consciousness requiring mechanical ventilation. Repeated MRI imaging revealed a significant increase in edema at the level of the basal nuclei and mesencephalon compatible with a Bickerstaff encephalitis. Clinical improvement was seen after initiation of high-dose methylprednisolone pulse therapy (1 g daily) and plasmapheresis for 5 consecutive days. Plasmapheresis was continued 3 days per week for 1 month, and corticosteroids were tapered over a period of 6 months. The results of diagnostic evaluation for autoantibodies in the context of autoimmune encephalitis were negative. A 16-year-old white girl experienced recurrent episodes of acute aseptic meningoencephalitis. Five months before the first episode, she developed purpura with histologic evidence of leukocytoclastic vasculitis on skin biopsy. During childhood, she had a history of recurrent otitis and mastoiditis. CH50 and AP50 were analyzed by using commercial assays according the manufacturer's instructions: the liposome assay on a SPAPLUS instrument (Binding Site, Birmingham, United Kingdom) for CH50, and the WIESLAB Complement System Alternative Pathway ELISA kit (Malmö, Sweden) for AP50. C3d was quantified in plasma EDTA by nephelometry as previously described E1Gehring T. Erdmann T. Rahm M. Graß C. Flatley A. O'Neill T.J. et al.MALT1 Phosphorylation controls activation of T lymphocytes and survival of ABC-DLBCL tumor cells.Cell Rep. 2019; 29: 873-888.e10Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar except the following modifications: the assay was performed on a BNII instrument (Siemens, Marburg, Germany), the C3d standard was obtained by incubation of plasma EDTA for 1 week at 37°C, and the anti-C3d antibody was obtained from DAKO (catalog No. A006). The detection limit, depending on the standard preparation, varies between 0.2 and 0.4 mg/dL. Molecular biology analysis was performed at the Institut de Pathologie et de Génetique asbl. A panel of genes involved in complement disorders was analyzed with a custom kit from Roche (SeqCap EZ). This analysis, which was based on capture of genes of interest followed by high-throughput sequencing (on Illumina MiSeq or NextSeq), allows detection of point mutations and large-size intragenic rearrangements (deletion/duplication) of the coding region. Canonic transcripts of the following genes were used for the design: ADAMTS13, C3, C5, CD46 (MCP), CFB, CFHR5, CFI, DGKE, MMACHC, PLG, and THBD. All the coding exons and intronic junctions from –14 to +6 were sequenced with a minimal coverage of 100×. A search for deletion/duplication in CFH, CFHR1, CFHR2, CFHR3, and CFHR5 was conducted by using multiplex ligation-dependent probe amplification (with a P236 kit from MRC Holland for research purposes).

Thrombotic thrombocytopenic purpura (TTP) is a rare and unpredictable disease with a high mortality rate (90%) if untreated. It results from systemic microvascular thrombosis and leads to profound thrombocytopenia, hemolytic anemia and organ failure of varying severity. However, macrovascular thrombosis has been described in very rare cases. Caplacizumab has emerged as a promising new drug for the management of TTP. We report the case of a patient with idiopathic refractory TTP treated with caplacizumab who developed thrombotic complications upon discontinuation of treatment.

BACKGROUND AND AIMS:Several factors have been reported to affect faecal calprotectin [FC] values, and significant variation in FC concentrations has been observed in inflammatory bowel disease [IBD] patients. We aimed to evaluate FC variability in IBD patients, and to assess the robustness of a single stool punch.METHODS:This is a single-centre observational case-control study. Disease activity was assessed using endoscopic and clinical activity scores, as well as C-reactive protein levels. Stool samples were collected twice within a 1 to 6 days interval, and FC was measured on punches and homogenates by fluorometric enzyme immunocapture assay.RESULTS:In all, 260 stool samples were collected from 120 patients. Intrastool variability was low, with an intraclass correlation coefficient for single measures between three punches from a single stool sample of 0.91, and median coefficient of variation [CV] of 17%. CV of two stool samples a few days apart [intra-individual variability] were significantly higher [p <0.01] with median CV of 36%. FC standard deviations correlated with mean FC levels either for intrastool or for intra-individual variability, with a Spearman's coefficient of rank correlation of 0.85 and 0.78, respectively [p <0.01]. Disease type, location, activity, and FC levels did not influence variability.CONCLUSIONS:A single stool punch is reliable for FC measurement, considering that intrastool variability is low. Intra-individual variability a few days apart is significantly higher. Therefore, decision-making strategies based on single measurements should consider this variability, to determine the minimum optimal variation to be achieved, rather than a cut-off, especially in high FC levels.

The aim of the present analysis was to evaluate sex-specific cut-off values of a high-sensitivity cardiac troponin T (hs-cTnT) assay and a high-sensitivity cardiac troponin I (hs-cTnI) assay in an emergency department setting.We retrospectively studied 1945 male and 1643 female emergency department patients in whom we had measured both Roche hs-cTnT and Abbott hs-cTnI routinely upon every troponin measurement request. We performed reclassification analyses of sex-specific thresholds versus sex-neutral thresholds of both assays. In addition, we performed sensitivity analyses to find those sex-specific cut-off values for the Roche hs-cTnT and the Abbott hs-cTnI assays with the lowest possible rate of discordant classifications by both assays.Compared with the classification by the sex-neutral thresholds (i.e., 14 ng/L for hs-cTnT and 26 ng/L for hs-cTnI), using sex-specific thresholds (i.e., 16 ng/L in males and 9 ng/L in females for hs-cTnT; and in 34 ng/L males and 16 ng/L in females for hs-cTnI) resulted in a total reclassification rate of 4% for hs-cTnT and 3% for hs-cTnI in male individuals, and of 11% and 6%, respectively, in female individuals. In our cohort, the sex-specific hs-cTnT cut-off values currently in use (i.e., 16 ng/L in males and 9 ng/L in females) were best matched to a hs-cTnI cut-off value of 11 ng/L in male and 5 ng/L in female individuals. Conversely, the sex-specific hs-cTnI cut-off values currently in use (i.e., 34 ng/L in males and 16 ng/L in females) were best matched to a hs-cTnT cut-off value of 49 ng/L in male and 24 ng/L in female individuals. These "harmonised" cut-off values reduced discordant classifications between both assays by 43–68% compared to using cut-off values currently in use.Especially in women, reclassification rates were high, when using sex specific versus sex-neutral thresholds. Best matching cut-off values for hs-cTnT and hs-cTnI were markedly different to those currently in use. These "harmonised" cut-off values minimised discordant classifications between both assays.

AbstractBackground:The measurement of complement components is clinically useful where a deficiency is suspected, or where excessive activation and consumption are present in disease. C2 deficiency carries an increased risk of developing systemic lupus erythematosus, recurrent infections and atherosclerosis. In this study, we have evaluated The Binding Site’s Human Complement C2 SPAPLUS®assay.Methods:Linearity was tested using 13 sample dilutions covering the standard measuring range. Within- and between-assay variabilities were calculated using five samples with different C2 concentrations. The correlation between C2 concentrations in EDTA-plasma and serum was assessed, as was the correlation between C2 measurements by the automated assay and radial immunodiffusion. C2 concentrations were compared with CH50 activity, and quantified in individuals with homozygous or heterozygous C2 deficiency, acquired angioedema and patients with chronic inflammatory conditions.Results:The assay was linear across the measuring range (3.8–42.3 mg/L). Intra- and interassay variability were 2.3%–3.8% and 0%–3.3%, respectively. Comparison between C2 measurements in EDTA-plasma and serum provided a strong correlation (p<0.0001, R2=0.82, slope 0.92), as did the correlation between the automated and radial immunodiffusion methods (p<0.0001, R2=0.89, slope 1.07). A positive correlation between C2 concentration and CH50 activity was demonstrated (p<0.0001, R2=0.48). Significant differences were observed between the median C2 concentrations obtained in healthy controls and the patient clinical samples, with homozygous C2-deficient patients giving below detectable results.Conclusions:This C2 SPAPLUS®assay allows the automated, rapid and precice quantification of complement C2 protein and could therefore be considered as a replacement for older, more time-consuming methods.

Objective: Determine the frequency of granulomatosis with polyangiitis (GPA) associated with non-identified ANCA (non-MPO, non-PR3 ANCA) and secondarily compare their clinic with GPA associated with MPO-positive or PR3-positive ANCA. Methods: In a monocentric retrospective observational study, clinical data of 398 patients with non-identified ANCA (titer of ANCA at least 1/80 by immunofluorescence on ethanol fixed PMN) was gathered over a period of 6 years. GPA patients from this population were compared with GPA patients with identified ANCA on the basis of clinical, biological, immunological and histological features. Results: The most common diseases associated with non-identified ANCA were inflammatory bowel diseases accounting for 17% of diseases. GPA accounted for only 1.8% of cases. There were no significant differences in terms of clinical and histological characteristics between GPA with non-identified ANCA and GPA with identified ANCA, but significantly higher CRP levels were observed in GPA patients with identified ANCA (p = 0.005). Localized disease (ear, nose and throat and/or lung involvement without any other systemic involvement) was more frequent in the group of GPA with nonidentified ANCA (p = 0.047) as compared to GPA with identified ANCA. This explains that the former group of patients was less frequently treated by cyclophosphamide than the latter (p = 0.016). Conclusion: GPA with non-MPO, non-PR3 ANCAs is relatively rare. Our study suggests that GPA with nonidentified ANCA differs from GPA with identified ANCA by the frequency of localized forms.