Complement-mediated Destruction Research Articles

The role of intravenous immunoglobulin in autoimmune diseases with dermatological implications.

Intravenous immunoglobulin (IVIG) therapy has emerged as a promising treatment option for various dermatological autoimmune diseases due to its immunomodulatory potential and low incidence of severe side effects. Despite its widespread use, the mechanism of action of IVIG in treating autoimmune diseases remains a topic of debate. IVIG is derived from the plasma fractionation of a large pool of donors, primarily consisting of the IgG isotype. Its main mechanisms of action involve neutralizing circulating autoantibodies via the F(ab')2 portion, inhibiting complement-mediated tissue destruction, and reducing the half-life of circulating autoantibodies through the Fc portion. This paper explores the growing utilization of IVIG as an off-label therapy in dermatological autoimmune or immune-mediated diseases, including autoimmune bullous disease (AIBS), dermatomyositis (DM), lupus erythematosus (LE), systemic sclerosis, scleromyxedema, Stevens-Johnson Syndrome (SJS), toxic epidermal necrolysis (TEN), pyoderma gangrenosum (PG), and necrobiotic xanthogranuloma (NXG). In this context, the sole large prospective, randomized trial was the 2022 ProDERM study, which demonstrate efficacy of IVIG in improving cutaneous manifestations among 95 DM patients compared to the placebo group. Moreover, although considered off-label, the use of IVIG is regarded as the first-line therapy for patients with scleromyxedema. As a first line of therapy, IVIg is only approved for Kawasaki Disease (KD) in the setting of vasculitis. The treatment in all other indications is mostly considered as adjuvant therapy only after failure of immunosuppressive therapy or in the presence of contraindications.

Legionella Pneumophila Presenting as a Rare Cause of Acute Thrombocytopenia: A Case Report and Review of Literature.

Legionella pneumophila can cause a wide spectrum of clinical manifestations, ranging from a mild flu-like illness to fulminant multi-organ involvement, characterised by severe pneumonia, diarrhoea, encephalopathy, shock, hepatic dysfunction and renal failure. Very rarely, it can be associated with haematologic conditions such as thrombotic thrombocytopenic purpura (TTP), haemolytic uraemic syndrome (HUS) and immune thrombocytopenic purpura (ITP). We report a rare case of L. pneumophila causing ITP and review previously published cases of thrombocytopenia associated with Legionellosis in the literature. A 53-year-old male presented with fevers, chills, a productive cough and severe haemoptysis. Blood work was remarkable for leukocytosis, severe thrombocytopenia and hyponatraemia. Computed tomography (CT) imaging showed left lower lobe lung consolidation, and a peripheral blood smear showed giant platelets consistent with ITP. Legionella urine antigen testing returned positive. He was treated with intravenous immunoglobin, steroid taper and a ten-day course of azithromycin, which led to normalisation of his platelet count and resolution of the pneumonia. L. pneumophila can lead to complement-mediated destruction of platelets resulting in ITP. Antibodies against L. pneumophila can also cross-react with the enzyme ADAMTS13, inhibiting its function and resulting in TTP and HUS. Additionally, L. pneumophila can infect vascular endothelial cells causing their death and stimulating release of von Willebrand factor (vWF) multimers into the bloodstream, promoting thrombosis and platelet consumption. It is important for internists to consider L. pneumophila in the differential for any patient presenting with pneumonia and severe thrombocytopenia. Earlier detection and intervention can lead to prevention of critical bleeding and better outcomes. Legionella pneumophila is rarely associated with different haematologic disorders resulting in severe bleeding diathesis as well as thrombosis.It is important for internists to consider Legionella pneumophila in the differential diagnosis for any patient presenting with pneumonia and severe thrombocytopenia.Earlier detection and intervention can lead to prevention of critical bleeding and better outcomes.

Paroxysmal Nocturnal Hemoglobinuria: Current Management, Unmet Needs, and Recommendations.

Paroxysmal nocturnal hemoglobinuria (PNH) is an ultra-rare, acquired clonal abnormality, which renders hematopoietic cells exquisitely sensitive to complement-mediated destruction. Classical features of PNH include intravascular hemolytic anemia, increased thrombotic risk, and manifestations related to end-organ damage (eg fatigue, chest pain, dyspnea, renal failure, and pulmonary hypertension). With supportive care alone, mortality rate of patients with PNH is approximately 35%. The anti-C5 monoclonal antibody, eculizumab, was the first targeted therapy approved for PNH, and led to improved hemoglobin, quality of life, reduced transfusion need, reduced thrombosis, and greater overall survival. More recently, therapeutics such as longer acting anti-C5 (ravulizumab) and anti-C3 (pegcetacoplan) medications have been approved, along with other novel therapeutics in late-stage clinical trials. Biosimilars of eculizumab are also now available. Proximal inhibitors (against C3, factor B, and factor D) have shown significant improvements in hemoglobin and transfusion-avoidance in patients who remain anemic despite C5 inhibition. Despite these novel therapies, some unmet challenges remain, including management of breakthrough hemolysis, clinically significant iatrogenic extravascular hemolysis, optimal management in pregnancy, and infection risk mitigation as new targets in the complement system are blocked. In addition, the use of self-administered subcutaneous and oral therapies raises concerns around treatment adherence and the risks of uncontrolled terminal complement. Given the ultra-rare nature of PNH, development is underway of a centralized international registry to capture and analyze the data as they mature for various new therapies and characterize the clinical challenges related to PNH management.

Unraveling the Clinical Spectrum: A Case Study of Classic and Aplasia-Related Paroxysmal Nocturnal Hemoglobinuria

Introduction: Paroxysmal nocturnal hemoglobinuria (PNH) is a rare nonmalignant clonal disorder of hematopoietic stem cells characterized by a phosphatidylinositol glycan protein A (PIGA) gene mutation. This mutation results in the deficiency of glycosylphosphatidylinositol (GPI)-anchored proteins, CD55 and CD59, on the cell surface, rendering red blood cells (RBC) susceptible to complement-mediated destruction. This leads to hemolytic anemia, thromboses, and bone marrow failure. Here we describe two cases of PNH presenting to a tertiary care center. Case 1 A 29-year-old male with a history of polysubstance use presented to the Emergency Department with a 2-day history of progressively worsening left lower quadrant abdominal pain. CT abdomen pelvis showed multiple renal infarcts and findings suggestive of pneumatosis intestinalis. A complete blood count (CBC) revealed pancytopenia with a leukocyte count (WBC) of 3 x 10 9/L, hemoglobin (Hb) of 94 g/L, and platelet count (Plt) of 48 x 10 9/L. Lactate dehydrogenase (LDH) was elevated (900 IU/L), haptoglobin level was low ( <8mg/dL), and direct antiglobulin test (DAT) was negative. Urine was positive for hemosiderin, and flow cytometry showed RBCs (6.7%), granulocytes (97.97%), and monocytes (89.02%) with PNH phenotype. Bone marrow biopsy (BMB) was negative for dysplasia and fibrosis with normal hematopoiesis but showed increased erythroid precursors. Enoxaparin was started for thromboses and levofloxacin for neutropenia. Treatment for PNH was initiated with a loading dose of ravulizumab and meningococcal vaccination. The patient was lost to follow-up after receiving the first maintenance dose. Case 2 An 81-year-old female with a medical history of hypertension and coronary artery disease was hospitalized for gradually worsening shortness of breath and fatigue ongoing for 2 months. She reported easy bruising but denied a history of obvious bleeding from any orifice. Physical exam was significant for petechial rash in her lower extremities. Labs revealed pancytopenia (WBC 1.44 x 10 9/L, Hb 68 g/L, Plt 8 x 10 9/L), low reticulocyte count, LDH of 276 IU/L, and negative DAT. BMB showed marked aplasia, and she became transfusion dependent. Subsequently, flow cytometry revealed PNH phenotype in 1.45% of RBCs, 54% granulocytes, and 54% monocytes. Eculizumab therapy was started for the diagnosis of PNH. However, her transfusion requirements did not decrease. She developed iron overload and showed positive conversion on DAT. Darbepoetin alfa and romiplostim were added. Her Hb stabilized at >70g/L in 3 years, following which she only required romiplostim to maintain Plt >20,000 ( Graph 1). After 5 years, she opted to discontinue all treatment. She is being followed up regularly and has since remained stable. Discussion The two cases above are reflective of how PNH can present in different ways, ranging from the classic triad of symptoms to those with subclinical findings associated with other underlying conditions, such as aplastic anemia or myelodysplastic syndrome. The introduction of complement inhibitors completely changed the natural history of PNH, as patients' median survival shifted from 15-20 years to align with age-matched controls. Indications for treatment include severe anemia, thrombosis, or PNH symptoms like dyspnea, fatigue, and painful paroxysms. Currently, eculizumab and ravulizumab are the only FDA-approved drugs for PNH treatment. Ravulizumab is non-inferior to eculizumab, with the added advantage of less frequent dosing (every 8 weeks versus 2 weeks). Regular monitoring for hemolysis is warranted with treatment. Many patients require long-term therapy, which continues to be a significant challenge, even with the spaced dosing of ravulizumab. Eculizumab protects from terminal complement-mediated damage preventing intravascular hemolysis. However, these cells are still deficient in CD55, making them vulnerable to opsonization by C3 and premature removal in the spleen. As a result, >50% of PNH patients receiving treatment show positive conversion on DAT, as seen in case 2. Heparin is preferred in acute thrombotic states. Most hematologists recommend anticoagulation till a few months after starting complement inhibition.

Severe Acute Kidney Injury in Children as a Rare Complication of Paroxysmal Cold Hemoglobinuria.

Paroxysmal cold hemoglobinuria (PCH) is a rare condition in childhood characterized by complement-mediated premature destruction of red blood cells. PCH is associated with intravascular hemolysis causing hemoglobinuria, which may result in acute kidney injury of various severity. We aimed to retrospectively analyze clinical and laboratory features of children with PCH-associated acute kidney injury received at tertiary Pediatric Hematology and Nephrology Center, University Motol Hospital, Prague, Czech Republic during the period 2016 to 2022. We present here 3 children with PCH-associated acute kidney failure requiring renal replacement therapy. We highlight the association of PCH with kidney disease. Renal parameters and urine examination should be regularly tested in all children with PCH.

More targets, more options, more awareness: Moving toward a personalized treatment approach in PNH

aroxysmal nocturnal hemoglobinuria (PNH) is an acquired disorder of hematopoietic stem cells that results in the complement-mediated destruction of red blood cells (RBCs). Although PNH is a rare disease, interest and awareness are growing as we develop further insights into the mechanisms of disease and possible therapeutic targets. At the recent annual meeting of the European Hematology Association, five sponsored symposia, three podium presentations, and over 30 posters and abstracts explored the latest data on PNH. This report will focus on the latest clinical data for established and emerging treatment options, and how these may eventually help clinicians to provide more individualized management for each patient with PNH.

Low copy numbers of complement C4 and C4A deficiency are risk factors for myositis, its subgroups and autoantibodies

BackgroundIdiopathic inflammatory myopathies (IIM) are a group of autoimmune diseases characterised by myositis-related autoantibodies plus infiltration of leucocytes into muscles and/or the skin, leading to the destruction of blood vessels...

Receptor clustering and pathogenic complement activation in myasthenia gravis depend on synergy between antibodies with multiple subunit specificities

Myasthenia gravis is an autoimmune disorder defined by muscle weakness and fatigability associated with antibodies against proteins of the neuromuscular junction (NMJ). The most common autoantibody target is the acetylcholine receptor (AChR). Three mechanisms have been postulated by which autoantibodies might interfere with neurotransmission: direct antagonism of the receptor, complement-mediated destruction of the postsynaptic membrane, and enhanced internalization of the receptor. It is very likely that more than one of these mechanisms act in parallel. Dissecting the mechanisms of autoantibody-mediated pathology requires patient-derived, monoclonal antibodies. Using membrane antigen capture activated cell sorting (MACACS), we isolated AChR-specific B cells from patients with myasthenia gravis, and produced six recombinant antibodies. All AChR-specific antibodies were hypermutated, including isotypes IgG1, IgG3, and IgG4, and recognized different subunits of the AChR. Despite clear binding, none of the individual antibodies showed significant antagonism of the AChR measured in an in vitro neuromuscular synapse model, or AChR-dependent complement activation, and they did not induce myasthenic signs in vivo. However, combinations of antibodies induced strong complement activation in vitro, and severe weakness in a passive transfer myasthenia gravis rat model, associated with NMJ destruction and complement activation in muscle. The strongest complement activation was mediated by combinations of antibodies targeting disparate subunits of the AChR, and such combinations also induced the formation of large clusters of AChR on the surface of live cells in vitro. We propose that synergy between antibodies of different epitope specificities is a fundamental feature of this disease, and possibly a general feature of complement-mediated autoimmune diseases. The importance of synergistic interaction between antibodies targeting different subunits of the receptor can explain the well-known discrepancy between serum anti-AChR titers and clinical severity, and has implications for therapeutic strategies currently under investigation.

The Role of Fc Gamma Receptors in Antibody-Mediated Rejection of Kidney Transplants.

For the past decades, complement activation and complement-mediated destruction of allograft cells were considered to play a central role in anti-HLA antibody-mediated rejection (AMR) of kidney transplants. However, also complement-independent mechanisms are relevant in the downstream immune activation induced by donor-specific antibodies, such as Fc-gamma receptor (FcγR)-mediated direct cellular activation. This article reviews the literature regarding FcγR involvement in AMR, and the potential contribution of FcγR gene polymorphisms to the risk for antibody mediated rejection of kidney transplants. There is large heterogeneity between the studies, both in the definition of the clinical phenotypes and in the technical aspects. The study populations were generally quite small, except for two larger study cohorts, which obviates drawing firm conclusions regarding the associations between AMR and specific FcγR polymorphisms. Although FcγR are central in the pathophysiology of AMR, it remains difficult to identify genetic risk factors for AMR in the recipient’s genome, independent of clinical risk factors, independent of the donor-recipient genetic mismatch, and in the presence of powerful immunosuppressive agents. There is a need for larger, multi-center studies with standardised methods and endpoints to identify potentially relevant FcγR gene polymorphisms that represent an increased risk for AMR after kidney transplantation.

P822: A PHASE 1 SINGLE ASCENDING DOSE STUDY OF CAN106, A LONG-ACTING ANTI-C5 COMPLEMENT MONOCLONAL ANTIBODY IN CLINICAL DEVELOPMENT FOR PNH AND OTHER COMPLEMENT-MEDIATED DISEASES

Background: Paroxysmal nocturnal hemoglobinuria (PNH) is a rare, acquired genetic disorder caused by clonal mutations in the PIGA gene. Deficiency of PIGA protein renders erythrocytes susceptible to complement-mediated destruction, causing intravascular and extravascular hemolysis, which can lead to anemia, fatigue, thromboembolism, major organ damage, and death. Despite the approval of one anti-C3 and two anti-C5 therapies for PNH, high treatment costs limit market access in many countries. CAN106 is a novel anti-C5 monoclonal antibody designed for optimal pH-dependent binding to C5 and enhanced binding to FcRN to increase intracellular recycling and prolong its half-life. Aims: This first-in-human Phase 1 study aimed to evaluate the single-dose safety and tolerability, pharmacokinetics, pharmacodynamics, and immunogenicity of CAN106. Methods: This was a randomized, double-blind, placebo-controlled, single-ascending-dose study. We enrolled 31 healthy adult subjects at a single site in Singapore who received CAN106 (mg/kg) or placebo at one of the following 6 doses (mg/kg) and ratios: (0.25, n=1:0; 0.75, n=1:0; 2, n=3:2; 4, n=6:2; 8, n=6:2 or 12, n=6:2). The primary endpoint was the incidence of adverse events overall and by intensity, seriousness, type, and relatedness to study drug. Secondary endpoints were the pharmacokinetic characterization of CAN106, the pharmacodynamic effects on free C5 (target engagement) and CH50 (serum hemolytic activity), and the incidence of anti-drug antibodies. The original study duration of 196 days was shortened to 112 days based upon the observed half-life of CAN106 and the return of CH50 to normal values. Results: The mean age of subjects was 34 years, and 97% were male. All 31 subjects completed the study. Seven CAN106 subjects experienced 20 treatment-emergent adverse events (TEAEs), and three placebo subjects experienced five TEAEs. Most TEAEs were mild and not related to CAN106, and all resolved without any sequelae. Three CAN106 subjects experienced seven drug-related TEAEs at the two highest doses. Most were mild, none was serious, and all resolved without any sequelae. These events included a moderate infusion-related reaction that resolved upon discontinuing treatment; mild dizziness; and mild elevations in ALT, AST, hemoglobin, hematocrit, and RBC. CAN106 exposure (Cmax and AUC) was dose-proportional, linear, and had low inter-subject variability (<20% CV). The terminal elimination half-life was approximately 32 days. CAN106 led to dose-dependent reductions in free C5 and CH50 within 24 hours. At the 8 and 12 mg/kg doses, all subjects showed >99% reduction in free C5 and >90% inhibition of CH50, with the latter sustained for 2 to 4 weeks. Two subjects in the 4 mg/kg cohort tested positive for anti-drug antibodies at single time points (pre-dose and Day 28). Summary/Conclusion: CAN106 was safe and well-tolerated at single doses up to 12 mg/kg in healthy adult subjects. The dose-exposure relationship was linear, and the 32-day half-life in healthy subjects was similar to that of ravulizumab, which is dosed every 8 weeks in patients, suggesting the potential for an extended dosing interval. CAN106 led to rapid, dose-dependent reductions in free C5 and CH50, and at the two highest doses, achieved complete and sustained complement blockade (>90% inhibition of CH50) for up to 4 weeks. These promising results support further clinical development of CAN106 in PNH and other complement-mediated diseases.

A Review of Disease Mechanisms and Current and Emerging Treatment Options for Generalized Myasthenia Gravis.

Generalized myasthenia gravis (gMG) is a disease resulting from impaired neuromuscular transmission due to presence of antibodies that block acetylcholine receptors. Autoantibodies to acetylcholine receptors directly impair the activity of ion channels that conduct nerve impulses, and cross-link acetylcholine receptors resulting in complement-mediated destruction, further worsening functional impairment and patient quality of life. Although current treatments for gMG include thymectomy, immunosuppressive therapies, intravenous immunoglobulins, and plasmapheresis, among other strategies, none of these treatments reduces all immunoglobulin G subfractions. However, with the novel neonatal Fc receptor antagonist efgartigimod, levels of all immunoglobulin G subfractions are reduced, addressing an important aspect of the underlying pathophysiology of gMG. Through this program, clinicians will consider novel mechanisms in gMG therapy, learn to counsel patients on the changing landscape of gMG therapies, and find ways to incorporate the latest efficacy and safety data into practice.

Current Opinions on the Clinical Utility of Ravulizumab for the Treatment of Paroxysmal Nocturnal Hemoglobinuria.

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare disorder of hematopoietic stem cells genetically defined by the acquisition of somatic mutations in the X-linked phosphatidylinositol glycan anchor biosynthesis, class A (PIGA) gene. PIGA is essential for the synthesis of glycosyl phosphatidylinositol (GPI) anchor proteins and its mutations result in a deficiency of such molecules on the membrane of blood cells derived from the mutant clone. In particular, the lack of the GPI-linked complement regulatory proteins CD55 and CD59 is responsible for the increased sensitivity of PNH erythrocytes to complement-mediated destruction. Indeed, the classical clinical picture of PNH includes signs and symptoms of intravascular hemolysis along with variable degrees of cytopenia and a strong tendency to thrombosis, hallmarks of the disease. Before the introduction of anti-complement inhibitors, PNH was characterized by a high mortality primarily due to thrombotic events. The approval of the terminal anti-complement inhibitor eculizumab in 2007 introduced a paradigm shift in the treatment of the disease with improvement of the chronic hemolytic process and dramatic reduction of the thrombotic rate. However, eculizumab has a relatively short half-life when considering a life-long treatment, with obvious consequences as to the quality of life of treated patients necessitating relatively frequent drug administrations. Moreover, up to 30% of PNH patients undergoing eculizumab therapy show a suboptimal response, continuing to require red cell transfusions because of extravascular hemolysis or breakthrough hemolytic episodes. In 2019, the FDA approved the second-generation C5 inhibitor ravulizumab, a long-lasting agent with a better control of disease manifestations. Herein, we discuss the use of ravulizumab in PNH, its differences with first-generation C5 inhibitors, the research evidence supporting the safety and efficacy of this drug and its impact on costs for health systems and quality of life of PNH patients.

P162 Incidence of neutropaenia and infections in lupus patients on immunosuppression

Abstract Background/Aims Neutropaenia occurs in 20-40% of patients with systemic lupus erythematosus (SLE) and is attributed to a combination of autoimmune and complement-mediated destruction of white cells, increased neutrophil apoptosis and medication side-effects. Commonly when patients become neutropaenic (&amp;lt;2x10^9/L), immunosuppressant doses are reduced or discontinued. However, studies have not consistently shown an increased risk of infection in SLE patients that suffer neutropaenia while taking immunosuppressants. This retrospective case note review aimed to examine the association between degree of neutropaenia and rates of infection in SLE patients. Methods Data was collected for 100 patients attending an SLE clinic in a tertiary London hospital between May 2019 and August 2020. Patients completed a questionnaire reporting the number and nature of infections they had suffered in the previous year, and whether antibiotics and/or hospitalisation was required. Clinical data was collected by retrospective review of electronic patient records including immunosuppressant therapy and neutrophil counts. Sustained neutropaenia was defined as a neutrophil count &amp;lt;2x10^9/L for &amp;gt;3 months, and severe neutropaenia defined as a neutrophil count of &amp;lt; 1x10^9/L). Results 95% of patients were female with a median age of 42 years (range 18-87). 55 (55%) of our patients were prescribed an immunosuppressant - 32 mycophenolate mofetil, 14 azathioprine, 4 rituximab, 4 methotrexate, 4 ciclosporin, 3 cyclophosphamide and 1 leflunomide. This cohort was more likely to develop neutropenia (n = 19, 35% vs n = 10, 22%, OR 1.8, p = 0.13), report one or more infections (n = 29, 53% vs n = 19, 42%), require antibiotics (n = 19, 35% vs n = 10, 22%) or inpatient treatment (13% vs 7%). However, immunosuppressed patients with neutropaenia &amp;gt;1x10^9/L (8/19) did not experience more infections than those without neutropaenia (21/36) (42% vs 58%, OR 0.5, p = 0.2). Chest, urine and upper respiratory tract were the most commonly reported infections. 29 (29%) patients had sustained neutropaenia, with only 2 (2%) patients experiencing severe neutropaenia - both latter patients experienced recurrent viral infections and were only taking hydroxychloroquine. Conclusion Immunosuppressive therapy was correlated with an increased prevalence in the absolute number of reported infections, antibiotic prescriptions and hospital admissions. Although there were more cases of infection and neutropenia in patients receiving immunosuppressants, rates of infection were not higher amongst neutropenic patients whose counts were above 1x10^9/L. Our data suggest that the increased risk of infection in our patients receiving immunosuppressants may be independent of the risk of neutropenia. Rates of infection, antibiotic use and hospital admission were not increased in patients with sustained neutropenia. There were few cases of severe neutropenia in our cohort, and in the absence of immunosuppressant use, this may have been driven by the SLE itself. Disclosure S. Manou-Stathopoulou: None. W. Hann: None. A. Pakozdi: None. R. Rajakariar: None. M. Lewis: None. A. Cove-Smith: None. D. Pyne: None.

A deep-sea pathogenic Bacillus subtilis isolate employs different strategies to escape the killing of teleost and murine complements

Bacillus subtilis subsp. subtilis G7 was isolated from a deep-sea hydrothermal vent and is pathogenic to pathogenic to fish (Japanese flounder) and mice. G7 is able to survive in host sera and phagocytes. In this study, we investigated the underlying mechanism of G7 serum resistance. We found that (i) the remaining complement activity was very low in G7-incubated flounder serum but high in G7-incubated mouse serum; (ii) cleaved C3 and C5 components were detected on flounder serum-incubated G7 but not on mouse serum-incubated G7; (iii) abundant uncleaved C5 was localized in G7-incubated mouse, but not flounder, serum; (iv) G7-incubated flounder, but not mouse, serum exhibited strong chemotactic activity; (v) pre-treatment with low-dose lysozyme abolished the serum resistance of G7. Hence, G7 activates flounder complement but is protected from complement-mediated destruction by its cell wall structure, while G7 prevents the activation of mouse complement. These results indicate that G7 employs different mechanisms to avoid the complement killing of different hosts.

Frequency and perturbations of various peripheral blood cell populations before and after eculizumab treatment in paroxysmal nocturnal hemoglobinuria

While red blood cells (RBCs) and granulocytes have been more studied, platelets and reticulocytes are not commonly used in paroxysmal nocturnal hemoglobinuria (PNH) flow-cytometry and less is known about susceptibility to complement-mediated destruction and effects of anti-complement therapy on these populations.We performed flow-cytometry of RBCs and granulocytes in 90 PNH patients and of platelets and reticulocytes in a subgroup (N = 36), to unveil perturbations of these populations during PNH disease course before and after anti-complement treatment.We found that platelets and reticulocytes were less sensitive to complement-mediated lysis than RBCs but not as resistant as granulocytes, as shown by mean sensitive fraction (difference in a given PNH population vs. PNH granulocyte clone size). In treated patients, reticulocytes, platelets, RBCs (with differences between type II and III) and granulocytes significantly increased post-treatment, confirming the role of PNH hematopoiesis within the context of anti-complement therapy. Moreover, we found that PNH platelet clone size reflects PNH granulocyte clone size. Finally, we established correlations between sensitive fraction of PNH cell-types and thrombosis.In sum, we applied a flow-cytometry panel for investigation of PNH peripheral blood populations' perturbations before and after eculizumab treatment to explore complement-sensitivity and kinetics of these cells during the disease course.

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Reduced red blood cell surface level of Factor H as a mechanism underlying paroxysmal nocturnal hemoglobinuria.

The absence of the cell-surface complement inhibitors CD55 and CD59 is considered the mechanism underlying the complement-mediated destruction of affected red blood cells (RBCs) in paroxysmal nocturnal hemoglobinuria (PNH) patients, but Factor H (FH), a fluid-phase complement inhibitor, has also been proposed to be involved. However, the status of FH on the PNH patient RBC surface is unclear and its precise role in PNH pathogenesis remains to be further defined. In this study, we identified significantly lower levels of surface-bound FH on the affected CD59- RBCs than on the unaffected CD59+ RBCs. Although this reduction in surface-bound FH on PNH RBCs was accompanied by decreased surface sialic acid levels, the enzymatic removal of sialic acids from these RBCs did not significantly affect the levels of surface-bound FH. We further observed higher surface levels of FH on the C3b/iC3b/high RBCs than on C3b/iC3blow RBCs within the affected PNH RBCs of patients treated with eculizumab. Finally, we determined that enhanced surface levels of FH on CD55/CD59-deficient RBCs from mice and PNH patients protected against complement-mediated hemolysis. Taken together, our results suggest that a reduced surface level of FH is another important mechanism underlying the pathogenesis of PNH.

A complement-mediated rat xenotransfusion model of platelet refractoriness

BackgroundPlatelet refractoriness remains a challenging clinical dilemma although significant advancements have been made in identifying human leukocyte antigen (HLA) matched or HLA compatible units. Antiplatelet antibodies are the major risk factor for immune-mediated platelet refractoriness, yet the role of antibody-initiated complement-mediated platelet destruction remains poorly understood. Study Design and MethodsHuman complement-mediated opsonization and killing of platelets was assayed ex vivo using antibody-sensitized human platelets incubated with complement-sufficient human sera. A new animal model of platelet refractoriness utilizing Wistar rats transfused with human platelets is described. ResultsHuman platelets sensitized with anti-platelet antibodies were rapidly opsonized with iC3b upon incubation in human sera. This opsonization could be completely blocked with a classical pathway complement inhibitor, PA-dPEG24. Complement activation decreased platelet viability, which was also reversible with complement inhibitor PA-dPEG24. A new rat model of platelet refractoriness was developed that demonstrated some platelet removal from the blood stream was complement mediated. ConclusionsComplement activation initiated by anti-platelet antibodies leads to complement opsonization and decreased platelet viability. A new rat model of platelet refractoriness was developed that adds a new tool for elucidating the mechanisms of platelet refractoriness.

Serum Complement Levels in Immune Thrombocytopenia: Characterization and Relation to Clinical Features

Background Complement may contribute to platelet destruction in immune thrombocytopenia (ITP), but serum complement levels of ITP patients are not well defined. This study characterized C3, C4, and CH50 levels from 108 ITP patients in comparison with 120 healthy subjects. Methods Results of complement testing performed using commercially available turbidimetric immunoassays were retrospectively analyzed. Mean complement levels in patients with ITP were compared with levels from a sample of 120 healthy subjects, and subgroups of ITP patients were compared. Regression analyses evaluated for relations between low complement levels and disease severity and response to ITP treatments. Results One hundred eight patients with ITP were included. Mean C3, C4, and CH50 were significantly lower in patients with ITP compared with healthy subjects, largely driven by the 32% of patients with ITP with substantial reductions in one or more assays. Patients requiring treatment had lower mean C4 (18.1 vs 23.1 mg/dL; P = .042) and CH50 (50.4 vs 63.0 mg/dL; P = .004). Mean C3 was higher in splenectomized versus nonsplenectomized patients (120.6 vs 101.0 mg/dL; P = .035). In multivariable analyses, reduced complement did not predict treatment response to corticosteroids, intravenous immunoglobulin, or thrombopoietin receptor agonists but low C4 levels did predict more severe ITP (relative to nonsevere disease, odds ratio for severe/refractory disease: 6.28; 95% confidence interval, 0.75-52.54; P = .090). Complement levels in patients with ITP were generally consistent over repeat measurements. Conclusions Complement levels are reduced in one-third of patients with ITP and are associated with more severe disease. Additional study is needed to evaluate if hypocomplementemia is predictive of response to emerging complement-directed therapies.

How Streptococcus suis serotype 2 attempts to avoid attack by host immune defenses.

Streptococcus suis (S. suis) type 2 (SS2) is an important zoonotic pathogen that causes swine streptococcosis, a widespread infectious disease that occurs in pig production areas worldwide and causes serious economic losses in the pork industry. Hosts recognize pathogen-associated molecular patterns (PAMPs) through pattern recognition receptors (PRRs) to activate both innate and acquired immune responses. However, S. suis has evolved multiple mechanisms to escape host defenses. Pathogenic proteins, such as enolase, double-component regulatory systems, factor H-combining proteins and other pathogenic and virulence factors, contribute to immune escape by evading host phagocytosis, reactive oxygen species (ROS), complement-mediated immune destruction, etc. SS2 can prevent neutrophil extracellular trap (NET) formation to avoid being trapped by porcine neutrophils and disintegrate host immunoglobulins via IgA1 hydrolases and IgM proteases. Currently, the pathogenesis of arthritis and meningitis caused by SS2 infection remains unclear, and further studies are necessary to elucidate it. Understanding immune evasion mechanisms after SS2 infection is important for developing high-efficiency vaccines and targeted drugs.