CD59 Deficiency Research Articles

BCX9930, an Oral Factor D Inhibitor, for the Potential Treatment of Alternative Pathway Mediated Diseases: Interim Results of a Phase 1 Study in Healthy Subjects

INTRODUCTION: Complement factor D is the rate-limiting enzyme for activation of the complement alternative pathway (AP). Targeting factor D is therefore an attractive therapeutic strategy for treatment of AP-mediated diseases including paroxysmal nocturnal hemoglobinuria (PNH), C3 glomerulopathy, and atypical hemolytic uremic syndrome. BCX9930 is a potent and selective orally bioavailable inhibitor of factor D in development for the treatment of PNH and other complement-mediated diseases. PNH is an acquired, chronic, non-malignant disorder of hematopoietic stem cells that is characterized by clonal deficiency of AP regulatory proteins CD55 and CD59, resulting in dysregulated AP amplification, opsonization, membrane attack complex (MAC) formation, and hemolysis. The approved intravenously (IV) administered C5 inhibitors eculizumab and ravulizumab inhibit MAC formation and intravascular hemolysis, but do not inhibit opsonization, leading to extravascular hemolysis and potentially incomplete treatment response. Inhibition of factor D is expected to prevent both intravascular and extravascular hemolysis in PNH, a distinct advantage over currently available parenteral therapies. METHODS: This abstract describes preliminary data in healthy subjects enrolled in the single ascending dose (SAD) and multiple ascending dose (MAD) evaluations of Study BCX9930-101 (NCT04330534), an ongoing first in human (FIH) study that includes randomized, double-blind, placebo-controlled evaluations of the safety and tolerability, pharmacokinetics (PK), and pharmacodynamics (PD) of BCX9930 in healthy subjects. Safety and tolerability are evaluated via clinical and laboratory monitoring. Plasma concentrations of BCX9930 are measured using a validated liquid chromatography-dual mass spectrometry (LC/MS/MS) assay; PD effects are assessed using multiple assays, including inhibition of AP activity in the AP-specific Wieslab (AP-W) and AP Hemolysis ([AP-H], lysis of rabbit red blood cells [RBCs] by human serum) assays. RESULTS: To date, 48 subjects have been enrolled into 6 SAD cohorts (n = 6 BCX9930, n = 2 placebo per cohort), with doses ranging from 10 to 1200 mg. Sixty subjects have been enrolled into 5 MAD cohorts (n = 10 BCX9930, n = 2 placebo per cohort), with doses ranging from 50 mg every 12 hours (Q12h) to 400 mg Q12h. The mean age of all enrolled subjects was 34.3 years and 50.9% were male. BCX9930 was safe and generally well-tolerated at all doses evaluated. There were no serious adverse events or dose-related safety signals. Mild headache was the most frequent treatment-emergent adverse event and showed no correlation with dose. A benign maculopapular exanthem attributed to study drug was observed following MAD administration. These events were mild or moderate in severity and resolved in a median of 5 days. BCX9930 exposure was approximately linear and dose-proportional across all evaluated SAD and MAD doses. Mean BCX9930 concentrations at 12 hours post dose were ≥ 8 to 10 times the factor D half-maximal effective concentration (EC50) for SAD doses ≥ 300 mg and for MAD doses ≥ 100 mg Q12h at steady-state. Rapid, dose-dependent AP suppression was observed following SAD and MAD, as measured by the AP-W and AP-H assays (Figure 1). AP suppression > 98% was observed in both assays at steady state for MAD doses ≥ 200 mg Q12h. Duration of complete suppression increased with dose and was ≥ 12 hours for MAD doses ≥ 200 mg Q12h. Doses ≥ 200 mg Q12h showed greater, more durable, and more consistent inhibition across subjects compared with doses ≤ 100 mg Q12h. PK/PD modeling of AP-H and AP-W demonstrates a clear concentration-response, with estimated EC50 values between 21.7 and 40.7 nM. These estimates are consistent with the in vitro IC50 for inhibition of proteolytic activity against the natural substrate C3bB of 28.1 nM. CONCLUSIONS: BCX9930 is a potent and selective orally bioavailable inhibitor of human complement factor D. In this preliminary analysis of an ongoing FIH study, BCX9930 displayed a promising safety and tolerability profile. Exposure was approximately linear and dose-proportional across a wide dose range, and complete and durable suppression of the AP was achieved. Together, these findings support further evaluation of BCX9930 in patients with PNH and other AP-mediated diseases. Disclosures Davidson: BioCryst Pharmaceuticals, Inc.:Current Employment.Mathis:BioCryst Pharmaceuticals, Inc.:Current Employment.Mair:Quotient Sciences:Current Employment.Gesty-Palmer:BioCryst Pharmaceuticals, Inc.:Current equity holder in publicly-traded company, Ended employment in the past 24 months.Cornpropst:BioCryst Pharmaceuticals, Inc.:Current Employment.Sheridan:BioCryst Pharmaceuticals, Inc.:Current Employment.Chen:BioCryst Pharmaceuticals, Inc.:Current Employment.Reynolds:BioCryst Pharmaceuticals, Inc.:Current Employment.Parker:BioCryst Pharmaceuticals, Inc.:Current Employment.Babu:BioCryst Pharmaceuticals, Inc.:Current Employment.

Dysphagia with Blood Disorder? an Unusual Presentation for Paroxysmal Nocturnal Hemoglobinuria

Paroxysmal Nocturnal Hemoglobinuria (PNH) is an acquired hemolytic disorder of the multipotent myeloid stem cells, due to underexpression of surface complement inhibitory proteins CD55 and CD59, affecting 0.13/100,000 patients per year. Besides anemia and thrombosis, PNH can also cause smooth muscle dystonias owing to various mechanisms in the body. Studies have described smooth muscle dystonias presenting as recurrent abdominal pain, lethargy and erectile dysfunction, but intermittent dysphagia is rare with PNH. We describe a case of dysphagia with negative work up as initial presentation of PNH. 16-year-old female with no significant past medical history presented to the clinic with 1 week of ongoing fatigue and difficulty swallowing. She never had any similar complaints in the past nor in her family. The dysphagia was progressively worsening, initially for solid, then with liquids, accompanied by nausea, vomiting and globus sensation in the middle chest. No prior psychiatric history, binging-purging behavior, chest pain, shortness of breath, heat/cold intolerance, weight changes, bladder or bowel dysfunction. She also denied any recent travels, allergies, transfusions, sick contacts, or over-the-counter medication use. Initial labwork showed Hb 11.2 g/dL, MCV 80 fL, WBC 9 x103 K/uL, and platelet 219,000 K/uL. Metabolic panel, troponin, BNP, ANA, rheumatoid factor, lyme serology, vitamin B12, folate and iron studies were within normal limits. Mentzer index <13, serum protein electrophoresis ruled out Thalassemia. LDH was elevated and Haptoglobin decreased. Direct and indirect Coomb's antibody test were negative. Peripheral smear showed normocytic normochromic anemia with normal morphology of RBCs. Flow cytometry showed lack of CD55 and CD59. Esophagogastroduodenoscopy, done for dysphagia, to rule out luminal obstruction was unremarkable. CT chest with contrast ruled out external compression on esophagus. Videofluoroscopic swallow study revealed segmental, dynamic obstruction to both solids and liquids in the lower third of the esophagus. Dysphagia was attributed to PNH and patient was started on eculizumab. She responded to the medication in 3 weeks, during which outpatient symptomatic therapy was given for her anemia along with soft semisolid diet. In PNH, the RBCs may have varying degree of CD59 and glycosyl phosphatidylinositol (GPI) anchor protein deficiency, with unaffected cells described as type I, partially affected cells called type II, and complete lack of the proteins deemed type III red cells. PNH red cell type III and type II red cells are 15 to 25 times and 3 to 5 times more sensitive to complement-mediated lysis than the type I normal red cells. This variability in the severity of the deficiency, as well as in the proportion of the cell population affected, defines the extent of the clinical manifestations of the disease. Approximately 40% of patients have a combination of types I, II and III PNH cells With hemolysis, after exceeding haptoglobin's binding capacity, the free hemoglobin binds any available nitrous oxide (NO) in the blood. Hemolysis also reduces endogenous NO generation by conversion of l-arginine, which is a component in NO generation, to ornithine, thereby further reducing the systemic availability of NO. NO is endogenous smooth muscle relaxor via activation of cGMP. Decreases in NO levels are hypothesized to cause smooth muscle contraction, manifesting as abdominal pain, erectile dysfunction and rarely, dysphagia in the lower two-third of the esophagus which is predominantly smooth muscle. These symptoms occur more commonly in patients with large PNH clone sizes and, therefore, higher hemolytic rates. In this case study, our focus was to recognize PNH among differentials for new onset dysphagia due to complement-mediated intravascular hemolysis causing gastrointestinal dysmotility. In doing so, invasive investigations may be avoided. Advance studies may be needed to completely understand the causes and correlate between PNH, intravascular hemolysis and dysphagia. Disclosures No relevant conflicts of interest to declare.

Recurrent angioedema, Guillain-Barr\xe9, and myelitis in a girl with systemic lupus erythematosus and CD59 deficiency syndrome

BackgroundCD59 deficiency is a congenital mutation disorder in complement pathway which can present with various manifestations.Case presentationHerein, we presented an adolescent 16-years-old girl with recurrent attacks of Guillain-Barre in early childhood and then recurrent attacks of angioedema, paresthesia, and myelitis. Finally, she presented with quadriplegia, malar rash, proteinuria, lymphopenia, and high titer of antinuclear antibody. So, the patient developed systemic lupus erythematosus. Furthermore, we performed whole exome sequencing which revealed homozygote mutations in CD59 for the patient and heterozygote one for her parents. CD flow cytometry showed less than 1 percent expression of CD59 on the surface of the patient’s peripheral blood cells confirming the disorder. So, she had CD59 deficiency. The patient’s episodes were managed with plasma exchanges, corticosteroids, Cyclophosphamide, and Mycophenolate Mofetil which induced and maintained remission.ConclusionCD59 deficiency can be presented with various clinical features such as neurologic, hematologic, dermatologic, and rheumatologic problems including systemic lupus erythematosus.

Complement factor CD59 deficiency significantly aggravates cholestasis liver injury in mice

Objective To investigate the role of complement factor CD59 in cholestatic liver injury in mouse bile duct ligation (BDL) model. Methods The cholestasis model of mice was induced by BDL. The relevant indexes were detected 3 days after operation. Results Increased alanine aminotransferase [ALT: (730.67±104.50) U/L vs. (311.33±52.47) U/L, t=8.784, P 0.05] and C5a [(284.43±80.88) μg/L vs. (212.95±13.53) μg/L, t=1.743, P>0.05] levels between the two BDL groups, suggesting that CD59 deficiency did not impact in vivo generation of C3 and C5 activation products. An increased membrane attack complex (MAC) deposition in CD59 deficiency animals could be monitored. Conclusion The present study proves a functional role of CD59 during cholestasis. CD59 deficiency leads to aggravated cholestatic liver injury by up-regulating the expression of MAC. CD59 appears to be an attractive target for future therapeutic intervention in chronic cholestatic liver disease. Key words: Cholestatic liver injury; CD59; Complement; Bile duct ligation; Membrane attack complex

CD55 Is Essential for CD103+ Dendritic Cell Tolerogenic Responses that Protect against Autoimmunity

Recent studies traced inflammatory bowel disease in some patients to deficiency of CD55 [decay-accelerating factor (DAF)], but the mechanism underlying the linkage remained unclear. Herein, we studied the importance of DAF in enabling processes that program tolerance in the gut and the eye, two immune-privileged sites where immunosuppressive responses are continuously elicited. Unlike oral feeding or ocular injection of ovalbumin in wild-type (WT) mice, which induced dominant immune tolerance, identical treatment of DAF-/- mice or DAF-/- to WT bone marrow chimeras did not. While 10% to 30% of mesenteric and submandibular lymph node CD4+ cells became robust T-regulatory cells (Tregs) in WT forkhead box P3 (Foxp3)-green fluorescent protein mice, few in either site became Tregs with little suppressor activity in DAF-/- Foxp3-green fluorescent protein mice. Phenotyping of CD103+ dendritic cells (DCs) from the ovalbumin-fed DAF-/- mice showed impaired expression of inducer of costimulation (ICOS) ligand, programmed death receptor 1-ligand 1 (PD1-L1), CxxxC chemokine receptor 1 (Cx3CR1), CCR7, and CCR9. Analyses of elicited DAF-/- Foxp3+ Tregs showed reduced expression of interferon regulatory factor 8 (IRF-8)/aldehyde dehydrogenase 1 family member A2 (Aldh1a2) and glycoprotein A repetitions predominant/latency-associated protein associated with Treg transforming growth factor-β production and presentation, as well as integrin β6/integrin β8 associated with Treg and CD103+ DC transforming growth factor-β release. Thus, DAF is required for the properties of CD103+ DCs and their naïve CD4+ cell partners that together program tolerance.

Paroxysmal nocturnal hemoglobinuria: the path to diagnosis

The aim of study was to describe the case of diagnosis of orphan disease — paroxysmal nocturnal hemoglobinuria in clinical practice of the therapist on an outpatient basis. Materials and methods . The patient Sh., 47 years, due to the gradual increase in the volume of the abdomen, the appearance of swelling of the legs, the increase in shortness of breath, yellowing of the skin, the appearance of rare but abundant nosebleeds appealed to 63polyclinic N. I. Pirogov City Clinical Hospital № 1. The patient was repeatedly hospitalized. During the diagnostic search, additional research methods were carried out: evaluation of laboratory data in dynamics; x-ray of the abdominal cavity; ultrasound examination of the abdominal cavity, kidneys and retroperitoneal space; computed tomography of the abdominal cavity; ultrasound examination of the pelvic organs; liver biopsy followed by microscopic examination of micro-drugs; samples for the diagnosis of autoimmune diseases; immunophenotyping of peripheral blood. The patient was consulted by a surgeon, hematologist. Results. In the course of difficult verification of such rare pathology, diagnostic and therapeutic concepts changed. The patient underwent symptomatic treatment: drainage of the abdominal cavity with evacuation of more than 14 liters of ascitic fluid; therapy with fresh frozen plasma; plasmapheresis. And only after positive clinical and laboratory dynamics during therapy with Eculizumab, immunophenotypic diagnosis was performed, that showed erythrocytes of type I (normal expression of CD59) — 1.2 %, type III (complete deficiency of CD59) — 49.5 %; monocytes with FLAER/CD14 deficiency — 83.4 %; granulocytes with FLAER/CD24 deficiency — 77.77 %. On the basis of these results, the diagnosis of paroxysmal nocturnal hemoglobinuria was established. Taking into account the severe course of the disease, according to vital indications, the patient continues therapy with Eculizumab at a maintenance dose of1200 mg every 14 (± 2) days. Conclusion . Timely highly qualified diagnosis, a modern approach in the treatment of this disease, provided a satisfactory outcome of the process in the patient with improved clinical condition and a favorable prognosis for health.

Eculizumab Is Safe and Effective as a Long-term Treatment for Protein-losing Enteropathy Due to CD55 Deficiency.

Loss of the complement inhibitor CD55 leads to a syndrome of early-onset protein-losing enteropathy (PLE), associated with intestinal lymphangiectasia and susceptibility to large-vein thrombosis. The in vitro and short-term treatment benefits of eculizumab (C5-inhibitor) therapy for CD55-deficiency have been previously demonstrated. Here we present the 18-months treatment outcomes for 3 CD55-deficiency patients with sustained therapeutic response. Three CD55-deficiency patients received off-label eculizumab treatment. Clinical and laboratory treatment outcomes included frequency and consistency of bowl movements, weight, patient/parent reports of overall well-being, and serum albumin and total protein levels. Membrane attack complex deposition on leukocytes was tested by flow cytometry, before and during eculizumab treatment. Marked clinical improvement was noted in all 3 patients with resolution of PLE manifestations, that is, diarrhea, edema, malabsorption, overall well-being, growth, and quality of life. In correlation with the clinical observations, we observed progress in all laboratory outcome parameters, including increase in albumin and total protein levels, and up to 80% reduction in membrane attack complex deposition on leukocytes (P < 0.001). The progress persisted over 18 months of treatment without any severe adverse events. CD55-deficiency patients present with early-onset diarrhea, edema, severe hypoalbuminemia, abdominal pain, and malnutrition. Targeted therapy with the terminal complement inhibitor eculizumab has positive clinical and laboratory outcomes in PLE related to CD55 loss-of-function mutations, previously a life-threatening condition. Our results demonstrate the potential of genetic diagnosis to guide tailored treatment, and underscore the significant role of the complement system in the intestine.

An update on the CD59 blood group system

This update of the CD59 blood group system (Weinstock C, Anliker M, von Zabern I. CD59: a long-known complement inhibitor has advanced to a blood group system. Immunohematology 2015;31:145-51) increases the number of reported patients with CD59 deficiency from 10 to 14. All of these 14 patients suffered from severe illness. Recently, a new variant allele was found in heterozygosity. Flow cytometry data suggest that this variant was expressed on the red blood cells of the propositus. Although additional alleles have been found, the CD59 system (International Society of Blood Transfusion system 35) continues to have one antigen.

CHAPLE syndrome uncovers the primary role of complement in a familial form of Waldmann's disease.

Primary intestinal lymphangiectasia (PIL) or Waldmann's disease was described in 1961 as an important cause of protein-losing enteropathy (PLE). PIL can be the sole finding in rare individuals or occur as part of a multisystemic genetic syndrome. Although genetic etiologies of many lymphatic dysplasia syndromes associated with PIL have been identified, the pathogenesis of isolated PIL (with no associated syndromic features) remains unknown. Familial cases and occurrence at birth suggest genetic etiologies in certain cases. Recently, CD55 deficiency with hyperactivation of complement, angiopathic thrombosis, and PLE (the CHAPLE syndrome) has been identified as a monogenic form of PIL. Surprisingly, loss of CD55, a key regulator of complement system leads to a predominantly gut condition. Similarly to other complement disorders, namely paroxysmal nocturnal and hemoglobinuria (PNH) and atypical hemolytic uremic syndrome (aHUS), CHAPLE disease involves pathogenic cross-activation of the coagulation system, predisposing individuals to severe thrombosis. The observation that complement system is overly active in CHAPLE disease introduced a novel concept into the management of PLE; anti-complement therapy. While CD55 deficiency constitutes a treatable subgroup in the larger pool of patients with isolated PIL, the etiology remains to be identified in the remaining patients with intact CD55.

Molecular pathogenesis of human CD59 deficiency.

ObjectiveTo characterize all 4 mutations described for CD59 congenital deficiency.MethodsThe 4 mutations, p.Cys64Tyr, p.Asp24Val, p.Asp24Valfs*, and p.Ala16Alafs*, were described in 13 individuals with CD59 malfunction. All 13 presented with recurrent Guillain-Barré syndrome or chronic inflammatory demyelinating polyneuropathy, recurrent strokes, and chronic hemolysis. Here, we track the molecular consequences of the 4 mutations and their effects on CD59 expression, localization, glycosylation, degradation, secretion, and function. Mutants were cloned and inserted into plasmids to analyze their expression, localization, and functionality.ResultsImmunolabeling of myc-tagged wild-type (WT) and mutant CD59 proteins revealed cell surface expression of p.Cys64Tyr and p.Asp24Val detected with the myc antibody, but no labeling by anti-CD59 antibodies. In contrast, frameshift mutants p.Asp24Valfs* and p.Ala16Alafs* were detected only intracellularly and did not reach the cell surface. Western blot analysis showed normal glycosylation but mutant-specific secretion patterns. All mutants significantly increased MAC-dependent cell lysis compared with WT. In contrast to CD59 knockout mice previously used to characterize phenotypic effects of CD59 perturbation, all 4 hCD59 mutations generate CD59 proteins that are expressed and may function intracellularly (4) or on the cell membrane (2). None of the 4 CD59 mutants are detected by known anti-CD59 antibodies, including the 2 variants present on the cell membrane. None of the 4 inhibits membrane attack complex (MAC) formation.ConclusionsAll 4 mutants generate nonfunctional CD59, 2 are expressed as cell surface proteins that may function in non–MAC-related interactions and 2 are expressed only intracellularly. Distinct secretion of soluble CD59 may have also a role in disease pathogenesis.

A Rare Cause of Elevated Liver Tests in a Pregnant Patient

Case: 26-year-old female at 22 weeks gestation with history of anemia presented with nausea, vomiting and sharp, epigastric, non-radiating abdominal pain for 4 days. She denied any bleeding. Surgical and medication history were notable for a previous dilation and curettage and a daily vitamin. Vital signs were normal, physical exam revealed pallor, gravid abdomen with epigastric tenderness to palpation. Hemoglobin was 6.8 g/dL and platelets 84 K/mcL. Liver panel showed AST 87 U/L, ALT 42 U/L and total bilirubin 3.2 mg/dL (direct 0.50 mg/dL). Reticulocytes were 8%, LDH 1142 U/L, haptoglobin undetectable with negative direct antiglobulin test. Peripheral smear showed macrocytosis, thrombocytopenia, anisopoikilocytosis, and no schistocytes. Acute viral hepatitis panel and parvovirus serologies were negative. Doppler ultrasound showed splenomegaly, normal fetus and liver with near occlusive thrombus of the middle hepatic vein with reversal of flow in the right portal vein. CT abdomen/pelvis revealed heterogeneous enhancement of liver with filling defects in multiple hepatic veins suggestive of thrombi. ADAMST13 level, protein C and S, antithrombin, glucose-6-phosphate dehydrogenase levels, factor V Leiden, Beta-2 glycoprotein, ANA, ASMA, SPEP and AMA were all negative. Endoscopy was negative for portal hypertensive gastropathy or varices. Duodenal biopsies were normal. Flow cytometry showed PNH clone percent of 84% in granulocytes, 68% in monocytes, and type III RBC (complete CD59 deficiency) of 8% confirming diagnosis of PNH. The patient was transfused, started on anticoagulation and eculizumab. Her anemia improved and she delivered a healthy, full-term baby. Discussion: PNH is characterized by a triad of intravascular hemolysis, thrombosis and bone marrow failure. Thrombosis occurring in atypical locations should prompt consideration of PNH. The hepatic vein is the most frequently involved vessel. PNH is associated with increased fetomaternal morbidity and mortality and thrombosis is the leading cause of death. Unexplained hemolytic anemia in a pregnant patient with concomitant abnormal liver tests may initially evoke diagnosis of HELLP (hemolysis, elevated liver enzymes, and low platelets), however, management of the two condition differs vastly. Anticoagulation is recommended for pregnant women with PNH thrombosis unless contradicted.Eculizumab reduces the risk of thrombosis. BMT is the only curative therapy for PNH with success rates of up to 100%.

CD59 is a potential biomarker of esophageal squamous cell carcinoma radioresistance by affecting DNA repair

Radiation therapy is an important treatment modality for esophageal cancer. However, acquisition of radioresistance ultimately results in esophageal cancer relapse. CD59, a membrane-bound complement regulatory protein, can transduce signals via a Src kinase in the lipid raft, thus playing a complement-independent role. However, the effect of CD59 on the esophageal cancer response to ionizing radiation remains unclear. In this study, we found that the expression level of CD59 was positively correlated with the radioresistance of esophageal cancer cell lines and clinical specimens. High CD59 expression indicated poor overall survival (OS) and disease-free survival (DFS) in esophageal squamous cell carcinoma (ESCC) patients who received radiotherapy. Genetic alteration of CD59 expression modulated the radiosensitivity of esophageal cancer cells to ionizing radiation. CD59 deficiency exacerbated DNA damage, hindered cell proliferation, and induced G2/M cell cycle arrest and cellular senescence, leading to an impaired DNA damage repair ability. In addition, CD59 deficiency almost completely reduced the phosphorylation of Src at Y416 despite ionizing radiation. A Src inhibitor saracatinib sensitized esophageal cancer cells to irradiation. Therefore, CD59 may be a potential biomarker for predicting the radioresistance of ESCC to radiotherapy.

Beyond the Role of CD55 as a Complement Component.

The complement is a part of the immune system that plays several roles in removing pathogens. Despite the importance of the complement system, the exact role of each component has been overlooked because the complement system was thought to be a nonspecific humoral immune mechanism that worked against pathogens. Decay-accelerating factor (DAF or CD55) is a known inhibitor of the complement system and has recently attracted substantial attention due to its role in various diseases, such as cancer, protein-losing enteropathy, and malaria. Some protein-losing enteropathy cases are caused by CD55 deficiency, which leads to complement hyperactivation, malabsorption, and angiopathic thrombosis. In addition, CD55 has been reported to be an essential host receptor for infection by the malaria parasite. Moreover, CD55 is a ligand of the seven-span transmembrane receptor CD97. Since CD55 is present in various cells, the functional role of CD55 has been expanded by showing that CD55 is associated with a variety of diseases, including cancer, malaria, protein-losing enteropathy, paroxysmal nocturnal hemoglobinuria, and autoimmune diseases. This review summarizes the current understanding of CD55 and the role of CD55 in these diseases. It also provides insight into the development of novel drugs for the diagnosis and treatment of diseases associated with CD55.

Diminished presentation of complement regulatory protein CD55 on red blood cells from patients with hereditary haemolytic anaemias

Hereditary haemolytic anaemias (HHA) encompass a heterogeneous group of anaemias characterized by decreased red blood cell survival. The aim of this study was to evaluate the status of red blood cell (RBC) surface molecules known or previously proposed to participate in preventing premature RBC clearance, analysing erythrocytes from patients with two types of HHA: hereditary spherocytosis (HS) and microcytosis. Relative binding of five monoclonal antibodies (mAbs), anti-CD55, anti-CD59, anti-CD44, anti-CD47 and anti-CD58, was evaluated in erythrocytes of patients with HS and hereditary microcytosis, using flow cytometry. The amount of CD55 protein was assessed by semi-quantitative Western blots densitometry analysis. The majority of both HS and microcytic patients demonstrated significant reduction of anti-CD55 binding by erythrocytes (average 23% and 19%, respectively, P<.001), with no concomitant anti-CD59-binding deficiency. Anti-CD44, anti-CD47 and anti-CD58 binding was within the healthy control range or was slightly decreased. This study provides evidence supporting the presence of erythrocytes deficient in CD55 presentation in HS and hereditary microcytosis. Moreover, deficiency of CD55 antigen presentation on RBC does not correlate with the amount of CD55 in RBC membrane. Further studies using molecular techniques will clarify the exact participation of CD55 deficiency in premature RBC clearance in HHA.

Decreased expression levels of complement regulator CD55 contribute to the development of bullous pemphigoid.

Bullous pemphigoid is a common autoimmune blistering disease of the elderly associated with autoantibody-mediated complement activation, and complement dysregulation is critical for its pathogenesis. As a crucial regulator of the complement system, CD55 has been widely studied in autoimmune diseases. Here, we investigated the involvement of CD55 in bullous pemphigoid, as little is known regarding its role in this disease. We found that CD55 levels were significantly lower in the lesions of patients with bullous pemphigoid (n = 8) compared to those in skin samples from healthy controls (n = 6). Interestingly, CD55 depletion in HaCaT human keratinocytes enhanced autoantibody-mediated complement activation. Moreover, complement activation was blocked by exogenous recombinant CD55 protein in both skin sections and keratinocytes exposed to pathogenic antibodies from patients with bullous pemphigoid. Notably, a significant increase in the expression of TNF-α and IFN-γ, administration of which downregulated CD55 levels in HaCaT cells, was observed in the sera of patients with bullous pemphigoid (n = 38) compared to that in healthy controls (n = 19). We found that ERK1/2 is involved in both TNF-α- and IFN-γ-induced CD55 downregulation. Thus, CD55 deficiency is a crucial factor in bullous pemphigoid pathogenesis, suggesting that increasing CD55 levels may exert a therapeutic effect.

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.)

Eculizumab treatment: stochastic occurrence of C3 binding to individual PNH erythrocytes

BackgroundC5 blockade by eculizumab prevents complement-mediated intravascular hemolysis in paroxysmal nocturnal hemoglobinuria (PNH). However, C3-bound PNH red blood cells (RBCs), arising in almost all treated patients, may undergo extravascular hemolysis reducing clinical benefits. Despite the uniform deficiency of CD55 and of CD59, there are always two distinct populations of PNH RBCs, with (C3+) and without (C3−) C3 binding.MethodsTo investigate this paradox, the phenomenon has been modeled in vitro by incubating RBCs from eculizumab untreated PNH patients with compatible sera containing eculizumab, and by assessing the C3 binding after activation of complement alternative pathway.ResultsWhen RBCs from untreated patients were exposed in vitro to activated complement in the context of C5-blockade, there was the prompt appearance of a distinct C3+ PNH RBC population whose size increased with time and also with the rate of complement activation. Eventually, all PNH RBCs become C3+ to the same extent, without differences between old and young (reticulocytes) PNH RBCs.ConclusionsThis study indicates that the distinct (C3+ and C3−) PNH RBC populations are not intrinsically different; rather, they result from a stochastic all-or-nothing phenomenon linked to the time-dependent cumulative probability of each individual PNH red cell to be exposed to levels of complement activation able to trigger C3 binding. These findings may envision novel approaches to reduce C3 opsonization and the subsequent extravascular hemolysis in PNH patients on eculizumab.

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.

Altered Expression Pattern of CD55 and CD59 on Red Blood Cells in Anemia of Chronic Kidney Disease

Objective: The aim of this study was to investigate the expression pattern of CD55 and CD59 on red blood cells (RBCs) in anemic chronic kidney disease (CKD) patients, and factors that might influence their expression. Subjects and Methods: Nighty-one adult anemic CKD patients and 80 healthy controls (HCs) were enrolled. Anemic CKD patients were divided into 3 subgroups based on receiving erythropoietin and renal replacement therapies. Flow cytometric analysis of CD55 and CD59 expression was performed on RBCs from blood samples obtained from CKD patients and HCs. Results: CD59 deficiency was significantly higher among CKD patients than HCs (n = 68, 74.7%, vs. n = 13, 16.3%, respectively; p < 0.001). The median proportions of CD55- and CD59-deficient RBCs in CKD patients were significantly higher compared to HCs (0.34 vs. 0.15, and 4.3 vs. 2.0, p < 0.001 and p < 0.001, respectively). The mean fluorescence intensity (MFI) of CD55 and CD59 expression was significantly lower in CKD patients compared to HCs (1.2 vs. 2.8, and 17.0 vs. 20.3, p < 0.04 and p < 0. 001, respectively). The hemoglobin level was inversely correlated with the proportions of CD55- and CD59-deficient RBCs (r = -0.37, p < 0.001, and r = -0.22, p < 0.02, respectively). The number of CD59-deficient patients was significantly different between the 3 subgroups of CKD patients (p = 0.001), and a significant difference was present in the MFI of CD55 and CD59 expression among the 3 subgroups (p = 0.04 and p = 0.03, respectively). Conclusion: The expression pattern of CD55 and CD59 on RBCs is altered in anemic CKD patients, which could play a role in the pathogenesis of anemia in CKD.

Clinico-Haematological Features of Paroxysmal Nocturnal Haemoglobinuria.

The aim of this study was to determine the frequency of various clinico-haematological features in patients suffering from paroxysmal nocturnal haemoglobinuria (PNH). It was an observational study carried out from October 2008 - January 2016. All the patients of PNH, diagnosed on the basis of clinical and laboratory findings and confirmed by CD55 and CD59 deficiency on red cells by means of flow cytometry, were included in the study. A total of 22 patients were diagnosed which included 18 (81.8%) males and 4 (18.1%) females. Median age was 27 years. Pallor, fever, fatigability and haemoglobinuria were the most common clinical features. Pancytopenia was seen in 13 (59.09%) and hypocellular marrow was found in 14 (63.6%) patients. One patient presented with Budd Chiari syndrome.