Abnormal Complement Activation Research Articles

Abnormal complement activation is a sign of long COVID

Abnormal complement activation is a sign of long COVID

The Change of Complement Deposition after Complement Inhibitor Treatment in Paroxysmal Nocturnal Hemoglobinuria Patients

Background: Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hematopoietic stem cell disease in which abnormal complement activation results in hemolysis and thromboembolic events (TEE). Complement inhibitors (CI) have been proved to be able to suppress intravascular hemolysis (IVH) and reduce the risk of TEE. Few studies were available on the changes of complement deposition after CI and their relationship with efficacy. Methods: PNH patients treated with full dose eculizumab, crovalimab or iptacopan at Peking Union Medical Colleague Hospital from Oct 2021 to Jul 2023 were enrolled.Patients' clinical information and blood samples were collected after 0, 1, 3, 6, 9 and 12 months of treatment. The deposition of C5b-9, C3, C4b and factor B (FB) on peripheral red blood cells (RBC), white blood cells (WBC) and platelets (PLT) was detected by double-color staining flow cytometric assay. The efficacy of each CI was correlated with complement deposition levels on three blood lineages. Results: 23 PNH patients were enrolled. Among them, 13 patients were treated with C5 inhibitors (4 with eculizumab and 9 with crovalimab), 10 were treated with iptacopan (FB inhibitor). For patients treated with eculizumab, C5b-9+/RBC and C5b-9+/WBC significantly decreased after 3, 6 and 12 months of treatment(P<0.05); however, C3+/WBC and C3+/PLT significantly increased after 12 months (P<0.05). For covalimab-treated patients, C5b-9+/RBC significantly decreased after 6 and 12 months (P<0.05), while C3+/RBC, C3+/WBC, C4b+/RBC and C4b+/WBC increased (P<0.05) after 6 months. For those treated with iptacopan, after 1, 3, 6, 9 and 12 months, the deposition of C5b-9 on all three blood lineages significantly decreased (P<0.05), meanwhile, C3 and FB on all three lineages decreased (P<0.05); moreover, C4b+/WBC and C4b+/PLT decreased after 3, 6, 9 and 12 months (P<0.05). C5b-9+/RBC and C5b-9+/WBC were significantly associated with hemoglobin improvement after eculizumab treatment (P=0.004 and 0.018, respectively). C5b-9+/RBC was positively correlated with bilirubin (P=0.023) and C3+/RBC was positively correlated with absolute reticulocyte counts (Ret, P=0.001) after crovalimab treatment. After iptacopan treatment, C3+/RBC was positively correlated with Ret (P=0.010), C4b+/RBC was positively correlated with Ret (P=0.032) and lactose dehydrogenase (LDH, P=0.003), and FB+/RBC was positively correlated with LDH. Conclusion: Complement inhibitors could effectively reduce C5b-9 deposition. Unlike C5 inhibitors, iptacopan also reduced the deposition of C3, C4b, and FB. C5b-9+/RBC may be correlated with the efficacy of inhibiting intravascular hemolysis. Upstream complement deposition is probably associated with breakthrough hemolysis or extravascular hemolysis. 【Key words】Paroxysmal nocturnal hemoglobinuria, complement inhibitor, complement deposition, flow cytometric assay, hemolysis

Complement factor D targeting protects endotheliopathy in organoid and monkey models of COVID-19.

COVID-19 is linked to endotheliopathy and coagulopathy, which can result in multi-organ failure. The mechanisms causing endothelial damage due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remain elusive. Here, we developed an infection-competent human vascular organoid from pluripotent stem cells for modeling endotheliopathy. Longitudinal serum proteome analysis identified aberrant complement signature in critically ill patients driven by the amplification cycle regulated by complement factor B and D (CFD). This deviant complement pattern initiates endothelial damage, neutrophil activation, and thrombosis specific to organoid-derived human blood vessels, as verified through intravital imaging. We examined a new long-acting, pH-sensitive (acid-switched) antibody targeting CFD. In both human and macaque COVID-19 models, this long-acting anti-CFD monoclonal antibody mitigated abnormal complement activation, protected endothelial cells, and curtailed the innate immune response post-viral exposure. Collectively, our findings suggest that the complement alternative pathway exacerbates endothelial injury and inflammation. This underscores the potential of CFD-targeted therapeutics against severe viral-induced inflammathrombotic outcomes.

C5aR antagonist inhibits LPS-induced inflammation in human gingival fibroblasts via NF-κB and MAPK signaling pathways.

Abnormal complement activation is associated with periodontitis. W54011 is a novel non-peptide C5aR antagonist (C5aRA) that exhibits favorable anti-inflammatory effects in various inflammatory models. However, whether W54011 inhibits periodontitis has not yet been fully elucidated. To address this, we have investigated the probable anti-inflammatory mechanism of W54011 in LPS-treated inflammation in human gingival fibroblasts (HGFs). HGFs were isolated from healthy gingival tissue samples using the tissue block method and were identified with immunofluorescence staining. The CCK8 assay and reverse transcription-PCR (RT-PCR) were used to select the optimal induction conditions for Lipopolysaccharide (LPS) and C5aRA (according to supplementary data S1, S2 and S3). The levels of inflammatory cytokines, C5aR, and the activation of NF-κB/MAPK signaling pathways were determined by RT-quantitative PCR (RT-qPCR) and Western blotting. Immunofluorescence results showed that vimentin and FSP-1 were positive in HGFs and Keratin was negative in HGFs. Immunofluorescence staining demonstrated that C5aRA inhibited LPS-stimulated nuclear translocation of p-p65. RT-qPCR and Western blotting showed that C5aRA reduced the expression of IL-1β, IL-6, TNF-α, C5aR, p-p65, p-IκBα, p-JNK, p-c-JUN, and TLR4 in LPS-induced HGFs. These findings suggested that C5aRA attenuated the release of inflammatory cytokines in LPS-induced HGFs by blocking the activation of the NF-κB and MAPK signaling pathways.

The Complement System Mediates COVID-19 Endothelial Cell Injury, Modifying Cell Permeability

Background Coronavirus Disease 2019 (COVID-19) is a (SARS-CoV-2)-mediated respiratory disease. The severity of COVID-19 has been classified as mild, moderate, severe and critical per the WHO-China Joint Mission. Among a total of 72 314 case records from the Chinese Center for Disease Control and Prevention, approximately 81% of COVID-19 cases were defined as mild, 14% of as severe, and 5% as critical. The overall hospital mortality of COVID-19 cases is approximately 15-20%, but it is up to 40-49% among critical cases requiring intensive care unit admission. The complement system, is a key component of the innate immune response, consists of more than 50 soluble and membrane-bound proteins that provide defense against microbes and mediate inflammatory responses. The activation of the complement system involves a series of enzymatic transformations, where C3b acts as a mediator. The final step of the complement cascade is the formation of a membrane attack complex (MAC, C5b-9) on the plasma membrane (PM) of the cells. Several pathologies including thrombotic, inflammatory, autoimmune and age-related diseases are associated with abnormal complement activation. There is evidence linking the activation of the complement system and endothelial injury to organ damage and complications that increase the risk of mortality in COVID-19. We hypothesize that endothelial cell injury resulting from complement activation contributes to COVID-19-associated vascular injury and organ damage. Aims To identify differences in complement activation associated with mild and severe forms of COVID-19 To determine the role of differential complement activation by COVID-19 patient sera in mediating endothelial cell damage. Methodology Clinical information, and sera from SARS-CoV-2 positive patients with mild and severe COVID-19 were obtained from the Canadian COVID-19 Prospective Cohort Study (CANCOV) to identify complement activation and endothelial cell (EC) injury. Complement activation was determined using a previously established protocol (Noris et al., 2017). Briefly, primary human umbilical vein endothelial cells (HUVECs) were treated with adenosine diphosphate (ADP), followed by treatment with healthy control or COVID-19 patient serum. To confirm the biological activity of complement, we measured the deposition of C3b and C5b-9 on the plasma membrane of HUVECs via immunofluorescence (IF) on cells exposed to healthy or patient sera. In addition, a permeability assay using a transwell model was used to measure the changes in permeability induced by healthy or patient sera via complement activation. Briefly, HUVECs were seeded on the top part of the insert, allowing them to form a monolayer. Monolayer integrity was assessed by imaging cells stained with Hoechst (nuclear stain) and phalloidin (cytoskeleton stain) on the day of the experiment, as well as before and after incubation with healthy or patient sera. Results We were able to detect complement activation/deposition when exposing the cells to COVID-19 sera. As for C3b deposition, cells exposed to patient sera showed no difference compared to those treated with sera from healthy donors. However, we observed increased C5b-9 deposition on the PM of cells treated with patient sera compared to those treated with sera from healthy donors, with further increased deposition when cells are treated with deceased patient sera. We observed a trend demonstrating increased permeability when cells are treated with deceased COVID-19 patient sera compared to healthy donors. Cellular permeability is significantly lower in those cells treated sera from patients with mild or severe COVID-19. Conclusion There is a differential biological activity of complement on endothelial cells depending on the severity of COVID-19, this complement activation leads to an increase in cell permeability. While more research needs to be done, these results contribute to a better understanding of the consequences of complement activation on endothelium in COVID-19.

Hemolytic uremic syndrome: An updated review

Hemolytic uremic syndrome (HUS) is a microangiopathic thrombotic disease. HUS is classified into typical, secondary, and atypical types. All types are characterized by thrombocytopenia, acute kidney impairment, and hemolysis. Infection with Shiga toxin from Escherichia coli causes typical HUS. Atypical HUS is frequently caused by abnormal complement activation via the alternative pathway because of gene mutations or autoantibodies synthesis. Secondary HUS accompanies post-transplantation, autoimmunity, cancer, etc. Endothelial cell injury initiates cells destruction stimulates procoagulant state, which activates platelet and thrombus generation, producing ischemic tissue injury.
 HUS pathogenesis is the result of an ineffective complement activation cycle that causes endothelial cells damage, activating platelet and thrombus formation. In some atypical HUS cases, interrupting the pathogenesis cycle of HUS by inhibiting complement activation might be beneficial, but not in those with gene mutations and patients with typical or secondary HUS. Hence, the pathogenesis of HUS types understanding is required; therefore, a comprehensive review of the differences and resemblances between the HUS types will be discussed and updated.

Ex Vivo Test for Measuring Complement Attack on Endothelial Cells: From Research to Bedside

As part of the innate immune system, the complement system plays a key role in defense against pathogens and in host cell homeostasis. This enzymatic cascade is rapidly triggered in the presence of activating surfaces. Physiologically, it is tightly regulated on host cells to avoid uncontrolled activation and self-damage. In cases of abnormal complement dysregulation/overactivation, the endothelium is one of the primary targets. Complement has gained momentum as a research interest in the last decade because its dysregulation has been implicated in the pathophysiology of many human diseases. Thus, it appears to be a promising candidate for therapeutic intervention. However, detecting abnormal complement activation is challenging. In many pathological conditions, complement activation occurs locally in tissues. Standard routine exploration of the plasma concentration of the complement components shows values in the normal range. The available tests to demonstrate such dysregulation with diagnostic, prognostic, and therapeutic implications are limited. There is a real need to develop tools to demonstrate the implications of complement in diseases and to explore the complex interplay between complement activation and regulation on human cells. The analysis of complement deposits on cultured endothelial cells incubated with pathologic human serum holds promise as a reference assay. This ex vivo assay most closely resembles the physiological context. It has been used to explore complement activation from sera of patients with atypical hemolytic uremic syndrome, malignant hypertension, elevated liver enzymes low platelet syndrome, sickle cell disease, pre-eclampsia, and others. In some cases, it is used to adjust the therapeutic regimen with a complement-blocking drug. Nevertheless, an international standard is lacking, and the mechanism by which complement is activated in this assay is not fully understood. Moreover, primary cell culture remains difficult to perform, which probably explains why no standardized or commercialized assay has been proposed. Here, we review the diseases for which endothelial assays have been applied. We also compare this test with others currently available to explore complement overactivation. Finally, we discuss the unanswered questions and challenges to overcome for validating the assays as a tool in routine clinical practice.

Réactions d’hypersensibilité au fer intraveineux : le point de vue de l’allergologue

Intravenous iron infusions rarely result in severe hypersensitivity reactions. The primary suspected hypersensitivity mechanism is an abnormal complement activation by non-IgE antibodies to the carbohydrate moieties stabilizing iron formulations. A major risk factor for hypersensitivity reactions is related to the infusion speed. Fishbane-like reactions usually resolve after pausing the infusion, which can be resumed under medical surveillance and at a lower infusion rate. Yet, anaphylactic reactions require emergency first aid and subsequent strict avoidance of intravenous iron. Desensitization protocols can be implemented in selected cases and under strict medical surveillance to reduce the risks of severe reactions upon re-exposure.

Species-specific accumulation of ceramides in cerebrospinal fluid from encephalomyeloradiculoneurpathy patients associated with peripheral complement activation: A pilot study

Glycolipids are now known to be rapidly converted to mediators for inflammatory reactions or to signaling molecules that control inflammatory events in the nervous system. The present study aimed to explore whether disturbed glycolipids metabolism in the nervous system is present in patients with a neuroinflammatory disorder, encephalo-myelo-radiculo-neuropathy (EMRN), because most EMRN patients have been reported to exhibit autoantibodies against neutral glycolipids. Although molecular pathogenesis of this disorder remains unknown, we tried to search the immunochemical abnormalities in this disorder. ELISA for activated peripheral C5 complement and mass spectrometry analysis of cerebrospinal fluid clearly disclosed a significant upregulation of active C5 complement, C5a levels in sera as well as a significant accumulation of species-specific ceramides but not sphingomyelin in cerebrospinal fluid from EMRN patients. Furthermore, we confirmed the occurrence of anti-neutral glycolipids antibodies in all EMRN patients.Thus, the present study might indicate the pathophysiology of this disorder is the dysregulation of glycolipids metabolism and abnormal production of autoantibodies against neutral glycolipids resulting in the abnormal complement activation, although molecular basis for these sphingolipids dysregulation and the occurrence of autoantibodies against glycolipids remains to be elucidated at present. The present study implicates a new therapeutic strategy employing anti-ceramide and/or anti-complement therapy for this disorder.

Role of the Host Genetic Susceptibility to 2009 Pandemic Influenza A H1N1.

Influenza A virus (IAV) is the most common infectious agent in humans, and infects approximately 10–20% of the world’s population, resulting in 3–5 million hospitalizations per year. A scientific literature search was performed using the PubMed database and the Medical Subject Headings (MeSH) “Influenza A H1N1” and “Genetic susceptibility”. Due to the amount of information and evidence about genetic susceptibility generated from the studies carried out in the last influenza A H1N1 pandemic, studies published between January 2009 to May 2020 were considered; 119 papers were found. Several pathways are involved in the host defense against IAV infection (innate immune response, pro-inflammatory cytokines, chemokines, complement activation, and HLA molecules participating in viral antigen presentation). On the other hand, single nucleotide polymorphisms (SNPs) are a type of variation involving the change of a single base pair that can mean that encoded proteins do not carry out their functions properly, allowing higher viral replication and abnormal host response to infection, such as a cytokine storm. Some of the most studied SNPs associated with IAV infection genetic susceptibility are located in the FCGR2A, C1QBP, CD55, and RPAIN genes, affecting host immune responses through abnormal complement activation. Also, SNPs in IFITM3 (which participates in endosomes and lysosomes fusion) represent some of the most critical polymorphisms associated with IAV infection, suggesting an ineffective virus clearance. Regarding inflammatory response genes, single nucleotide variants in IL1B, TNF, LTA IL17A, IL8, IL6, IRAK2, PIK3CG, and HLA complex are associated with altered phenotype in pro-inflammatory molecules, participating in IAV infection and the severest form of the disease.

Detection of Complement Factor B Autoantibodies by ELISA.

Antibodies to autoantigens are implicated in a large number of diseases. Such autoantibodies may cause pathological activation of complement, an ancient humoral recognition and effector system of innate immunity; in addition, complement components or regulators may be target of autoantibodies and cause abnormal complement activation or function. Autoantibodies to complement proteins are in particular involved in kidney diseases. Those binding to complement convertase enzymes can cause enhanced stability of convertases and their increased resistance to regulation, thus promoting complement turnover. Here, we describe an ELISA method to detect factor B autoantibodies that bind to and stabilize the alternative complement pathway C3 convertase enzyme, C3bBb.

Neuropsychiatric manifestations associated with systemic lupus erythematosus: A case report and selective review of literature

Introduction: Systemic lupus erythematosus (SLE) is an autoimmune condition affecting multiple organ systems including central nervous system. Its aetiology lies in abnormal antibody formation and complement activation. Neuropsychiatric manifestations are known to occur in patients with SLE, and vary from psychosis, depression, anxiety to cognitive impairment. Here we report a case of depression with psychotic symptoms and suicidal ideations associated with SLE. Case Summary: We present the case of a 30-year-old female who presented with irritability, crying spells, feeling of guilt, referential and persecutory ideations along with suicidal ideations. She was diagnosed with severe depression with psychotic symptoms associated with SLE. She improved over a period of 6 weeks with Quetiapine 300mg along with symptomatic management of underlying medical condition. Conclusions: Depression with psychotic symptoms is not an uncommon presentation of SLE. It needs comprehensive management and multidisciplinary approach. Hence physician has to be sensitized regarding neuropsychiatric manifestations of SLE for improving the morbidity and mortality associated with underlying depression. Keywords: SLE-Depression-Psychosis.

Parallel epidemics, or nearly so: Certainties and uncertainties about SARS-CoV-2 in Italy

Parallel epidemics, or nearly so: Certainties and uncertainties about SARS-CoV-2 in Italy

A Single-Domain Antibody Targeting Complement Component C5 Acts as a Selective Inhibitor of the Terminal Pathway of the Complement System and Thus Functionally Mimicks the C-Terminal Domain of the Staphylococcus aureus SSL7 Protein.

The complement system is an efficient anti-microbial effector mechanism. On the other hand abnormal complement activation is involved in the pathogenesis of multiple inflammatory and hemolytic diseases. As general inhibition of the complement system may jeopardize patient health due to increased susceptibility to infections, the development of pathway-specific complement therapeutics has been a long-lasting goal over the last decades. In particular, pathogen mimicry has been considered as a promising approach for the design of selective anti-complement drugs. The C-terminal domain of staphylococcal superantigen-like protein 7 (SSL7), a protein secreted by Staphylococcus aureus, was recently found to be a specific inhibitor of the terminal pathway of the complement system, providing selective inhibition of cell lysis mediated by the membrane attack complex (MAC). We describe here the selection by phage display of a humanized single-domain antibody (sdAb) mimicking the C-terminal domain of SSL7. The antibody, called sdAb_E4, binds complement C5 with an affinity in the low micromolar range. Furthermore, sdAb_E4 induces selective inhibition of MAC-mediated lysis, allowing inhibition of red blood cell hemolysis and inhibition of complement deposition on apopto-necrotic cells, while maintaining efficient bactericidal activity of the complement terminal pathway. Finally, we present preliminary results indicating that sdAb_E4 may also be efficient in inhibiting hemolysis of erythrocytes from patients with paroxysmal nocturnal hemoglobinuria. Our data provide a proof of concept for the design of a selective MAC inhibitor capable of retaining complement bacteriolytic activity and this study opens up promising perspectives for the development of an sdAb_E4-derived therapeutics with application in the treatment of complement-mediated hemolytic disorders.

Guidelines for biomarkers in autoimmune rheumatic diseases - evidence based analysis.

Autoimmune rheumatic diseases are characterised by an abnormal immune system response, complement activation, cytokines dysregulation and inflammation. In last years, despite many progresses in managing these patients, it has been shown that clinical remission is reached in less than 50% of patients and a personalised and tailored therapeutic approach is still lacking resulting in a significant gap between guidelines and real-world practice. In this context, the need for biomarkers facilitating early diagnosis and profiling those individuals at the highest risk for a poor outcome has become of crucial interest. A biomarker generally refers to a measured characteristic which may be used as an indicator of some biological state or condition. Three different types of medical biomarkers has been suggested: i. mechanistic markers; ii. clinical disease markers; iii. therapeutic markers. A combination of biomarkers from these different groups could be used for an ideal more accurate diagnosis and treatment. However, although a growing body of evidence is focused on improving biomarkers, a significant amount of this information is not integrated on standard clinical care. The overarching aim of this work was to clarify the meaning of specific biomarkers during autoimmune diseases; their possible role in confirming diagnosis, predicting outcome and suggesting specific treatments.

Extra-renal manifestations of atypical hemolytic uremic syndrome.

Atypical hemolytic uremic syndrome (aHUS) is a rare and complex disease resulting from abnormal alternative complement activation with a wide range of clinical presentations. Extra-renal manifestations of aHUS can involve many organ systems, including the peripheral and central nervous, gastrointestinal, cardiovascular, integumentary, pulmonary, as well as the eye. While some of these extra-renal manifestations occur in the acute phase of aHUS, some can also occur as long-term sequelae of unopposed complement activation. Extra-renal symptoms are observed in approximately 20% of patients with aHUS, with the incidence of specific organ system complications ranging from a few case reports to 50% of described patients. Careful monitoring for extra-renal involvement is critical in patients with aHUS, as prompt evaluation and management may decrease the risk of high morbidity and mortality associated with aHUS.

Functional analyses of rare genetic variants in complement component C9 identified in patients with age-related macular degeneration

Age-related macular degeneration (AMD) is a progressive disease of the central retina and the leading cause of irreversible vision loss in the western world. The involvement of abnormal complement activation in AMD has been suggested by association of variants in genes encoding complement proteins with disease development. A low-frequency variant (p.P167S) in the complement component C9 (C9) gene was recently shown to be highly associated with AMD; however, its functional outcome remains largely unexplored. In this study, we reveal five novel rare genetic variants (p.M45L, p.F62S, p.G126R, p.T170I and p.A529T) in C9 in AMD patients, and evaluate their functional effects in vitro together with the previously identified (p.R118W and p.P167S) C9 variants. Our results demonstrate that the concentration of C9 is significantly elevated in patients' sera carrying the p.M45L, p.F62S, p.P167S and p.A529T variants compared with non-carrier controls. However, no difference can be observed in soluble terminal complement complex levels between the carrier and non-carrier groups. Comparing the polymerization of the C9 variants we reveal that the p.P167S mutant spontaneously aggregates, while the other mutant proteins (except for C9 p.A529T) fail to polymerize in the presence of zinc. Altered polymerization of the p.F62S and p.P167S proteins associated with decreased lysis of sheep erythrocytes and adult retinal pigment epithelial-19 cells by carriers' sera. Our data suggest that the analyzed C9 variants affect only the secretion and polymerization of C9, without influencing its classical lytic activity. Future studies need to be performed to understand the implications of the altered polymerization of C9 in AMD pathology.

Abnormal Complement Activation and Inflammation in the Pathogenesis of Retinopathy of Prematurity.

Retinopathy of prematurity (ROP) is a neurovascular complication in preterm babies, leading to severe visual impairment, but the underlying mechanisms are yet unclear. The present study aimed at unraveling the molecular mechanisms underlying the pathogenesis of ROP. A comprehensive screening of candidate genes in preterms with ROP (n = 189) and no-ROP (n = 167) was undertaken to identify variants conferring disease susceptibility. Allele and genotype frequencies, linkage disequilibrium and haplotypes were analyzed to identify the ROP-associated variants. Variants in CFH (p = 2.94 × 10−7), CFB (p = 1.71 × 10−5), FBLN5 (p = 9.2 × 10−4), CETP (p = 2.99 × 10−5), and CXCR4 (p = 1.32 × 10−8) genes exhibited significant associations with ROP. Further, a quantitative assessment of 27 candidate proteins and cytokines in the vitreous and tear samples of babies with severe ROP (n = 30) and congenital cataract (n = 30) was undertaken by multiplex bead arrays and further validated by western blotting and zymography. Significant elevation and activation of MMP9 (p = 0.038), CFH (p = 2.24 × 10−5), C3 (p = 0.05), C4 (p = 0.001), IL-1ra (p = 0.0019), vascular endothelial growth factor (VEGF) (p = 0.0027), and G-CSF (p = 0.0099) proteins were observed in the vitreous of ROP babies suggesting an increased inflammation under hypoxic condition. Along with inflammatory markers, activated macrophage/microglia were also detected in the vitreous of ROP babies that secreted complement component C3, VEGF, IL-1ra, and MMP-9 under hypoxic stress in a cell culture model. Increased expression of the inflammatory markers like the IL-1ra (p = 0.014), MMP2 (p = 0.0085), and MMP-9 (p = 0.03) in the tears of babies at different stages of ROP further demonstrated their potential role in disease progression. Based on these findings, we conclude that increased complement activation in the retina/vitreous in turn activated microglia leading to increased inflammation. A quantitative assessment of inflammatory markers in tears could help in early prediction of ROP progression and facilitate effective management of the disease, thereby preventing visual impairment.

CD55 Deficiency, Early-Onset Protein-Losing Enteropathy, and Thrombosis

BackgroundStudies of monogenic gastrointestinal diseases have revealed molecular pathways critical to gut homeostasis and enabled the development of targeted therapies.MethodsWe studied 11 patients with abdominal pain and diarrhea caused by early-onset protein-losing enteropathy with primary intestinal lymphangiectasia, edema due to hypoproteinemia, malabsorption, and less frequently, bowel inflammation, recurrent infections, and angiopathic thromboembolic disease; the disorder followed an autosomal recessive pattern of inheritance. Whole-exome sequencing was performed to identify gene variants. We evaluated the function of CD55 in patients’ cells, which we confirmed by means of exogenous induction of expression of CD55.ResultsWe identified homozygous loss-of-function mutations in the gene encoding CD55 (decay-accelerating factor), which lead to loss of protein expression. Patients’ T lymphocytes showed increased complement activation causing surface deposition of complement and the generation of soluble C5a. Costimulatory function and cytokine modulation by CD55 were defective. Genetic reconstitution of CD55 or treatment with a complement-inhibitory therapeutic antibody reversed abnormal complement activation.ConclusionsCD55 deficiency with hyperactivation of complement, angiopathic thrombosis, and protein-losing enteropathy (the CHAPLE syndrome) is caused by abnormal complement activation due to biallelic loss-of-function mutations in CD55. (Funded by the National Institute of Allergy and Infectious Diseases and others.)

Inherited CD55 Deficiency in Patients with Early Onset Protein-Losing Enteropathy and Thrombosis

Abstract Studies of monogenic gastrointestinal diseases have revealed molecular pathways crucial for gut homeostasis and enabled the development of targeted therapies. We studied 11 patients with abdominal pain and diarrhea caused by protein-losing enteropathy with primary intestinal lymphangiectasia, hypoproteinemia, malabsorption, bowel inflammation, recurrent infections, and angiopathic thromboembolic disease. DNA sequencing identified autosomal recessive mutations in the gene encoding CD55/Decay accelerating factor, leading to loss of protein expression. CD55 regulatory function including complement deposition, costimulation, and anaphylatoxin responses were assessed by shRNA and CRISPR-mediated gene suppression and rescue of CD55 expression using lentiviruses. In all patients, Patient T lymphocytes and CD55-deficient cell lines displayed uncontrolled complement activation causing C3d deposition and generation of C5a. Genetic reconstitution of CD55 or treatment with a complement-inhibitory therapeutic antibody normalized complement activation. Patient T lymphocytes produced increased tumor necrosis factor, which was associated with a decreased ratio of the anti-coagulatory protein thrombomodulin to the pro-coagulatory protein tissue factor on endothelial cells. Additionally, CD55 costimulation by CD97 and contingent production of interleukin-10 were defective in patient T lymphocytes. Altogether, CD55 deficiency with hyperactivation of complement, angiopathic thrombosis, and PLE (CHAPLE) disease is caused by abnormal complement activation due to autosomal recessive loss-of-function mutations in CD55, and may be effectively treated with complement-inhibitory therapeutics.