C5b-9 Membrane Attack Complex Research Articles

Potential role of CTSS in AMDImmune modulatory and anti-angiogenic effects of cathepsin S knockdown in ARPE-19 cells

We aimed to determine the role of cathepsin S (CTSS) in modulating oxidative stress-induced immune and inflammatory reactions and angiogenesis in age-related macular degeneration.Human retinal pigment epithelium cells line ARPE-19 (immature) were maintained and treated with H2O2. The expression of CTSS, inflammatory cytokines, and complement factors induced by oxidative stress was compared between cells incubated without (control) and with CTSS knockdown (using small interfering ribonucleic acid; siRNA). To evaluate the role of CTSS in angiogenesis, we assayed tube formation using human umbilical vein endothelial cells and conditioned medium from ARPE-19 cells. We also used a mouse model of laser-induced choroidal neovascularization.CTSS levels were higher in ARPE-19 cells treated with H2O2 than in control cells. Oxidative stress-induced CTSS resulted in significantly elevated transcription of nuclear factor kappa B-dependent inflammatory cytokines, complement factors C3a and C5a, membrane attack complex (C5b-9), and C3a and C5a receptors. siRNA-mediated knockdown of CTSS reduced the number of inflammatory signals. Furthermore, oxidative stress-induced CTSS regulated the expression of peroxisome proliferator-activated receptor γ and vascular endothelial growth factor A/Akt serine/threonine kinase family signaling, which led to angiogenesis. Tube formation assays and mouse models of choroidal neovascularization revealed that CTSS knockdown ameliorated angiogenesis in vitro and in vivo.The present findings suggest that CTSS modulates the complement pathway, inflammatory reactions, and neovascularization, and that CTSS knockdown induces potent immunomodulatory effects. Hence, it could be a promising target for the prevention and treatment of early- and late-stage age-related macular degeneration.

#1021 A phase 3 randomized controlled trial of ravulizumab in adult patients with IgA nephropathy

Abstract Background and Aims The pathogenesis of IgA nephropathy (IgAN) includes the deposition of immune complexes in the kidney and activation of the complement system. Complement components, including C5a (anaphylatoxin) and C5b-9 (membrane attack complex), are generated leading to mesangial inflammation and glomerular injury [1]. Ravulizumab, a humanized monoclonal antibody, is a long-acting complement C5 inhibitor that provides immediate, complete, and sustained inhibition of the terminal complement pathway, thereby targeting a key component of the pathophysiology of IgAN. A phase 2 study of ravulizumab (NCT04564339) demonstrated a rapid effect and significant reduction in proteinuria compared with placebo at week 26 (change from baseline in 24-hour urine protein to creatinine ratio [UPCR] –40.3% vs –10.9%), and treatment was well-tolerated [2]. This abstract describes the study design of the planned phase 3 trial of ravulizumab in adult patients with IgAN. Method This phase 3, randomized, double-blind, placebo-controlled trial (I CAN) will evaluate the efficacy and safety of ravulizumab (IV; weight-based dosing Q8W) in adults with IgAN. Eligibility criteria include a diagnosis of primary IgAN based on kidney biopsy at any point, estimated glomerular filtration rate (eGFR) ≥30 mL/min/1.73 m2 at screening, and UPCR ≥0.75 g/g or urine protein ≥1.0 g/day (Table 1). The study consists of a 6-week screening period, followed by randomization (1:1) of participants to ravulizumab or placebo and a 2-year blinded treatment period (Fig. 1); all patients receive a stable and maximally tolerated dose of renin-angiotensin system inhibitor therapy. Approximately 450 patients will be randomized, stratified by proteinuria (<2 g/g vs ≥2 g/g), eGFR (<45 vs ≥45 mL/min/1.73 m2), and SGLT2i use (yes/no). The co-primary endpoints are change from baseline to week 34 in 24-hour UPCR evaluated on the log scale and the annualized total eGFR slope over 106 weeks. The sample size for the proteinuria endpoint of n = 204 will provide ∼90% power to detect a 35% relative treatment effect in UPCR at week 34 at a one-sided significance level of 0.005. The total sample size of n = 450 for the eGFR endpoint will provide ∼90% power to detect a difference in total annualized eGFR slope of 2.2 mL/min/1.73 m2 at week 106 at a one-sided significance level of 0.025. An open-label exploratory cohort (n = 20) with eGFR 20-29 mL/min/1.73 m2 and with kidney biopsy within 6 months of or during screening will be enrolled and analyzed separately from the main cohort. At completion of the blinded treatment period, all study participants will have the option to enter an open-label ravulizumab access period for up to 2 years. Results The co-primary endpoints are the change in 24-hour UPCR at week 34 and the annualized total eGFR slope over 106 wks. Secondary and exploratory endpoints include change in 24-hour UPCR at weeks 10, 26, 50, and 106, a composite kidney failure endpoint over 106 weeks, reduction in 24-hour UPCR ≥50% from baseline, time to UPCR <1 g/g and <0.3 g/g, and patient reported outcomes on fatigue, quality of life, and work-related productivity. Safety, tolerability, immunogenicity, pharmacokinetics, and pharmacodynamics will be evaluated. Conclusion Through immediate, complete, and sustained inhibition of C5, ravulizumab targets the pathophysiology of IgAN and has the potential to provide a disease modifying treatment option. This pivotal phase 3 study (I CAN) will evaluate the efficacy of ravulizumab in patients with IgAN who are at high risk of disease progression. The trial will evaluate effects on proteinuria and eGFR that may translate to long term kidney function preservation and provide a rapidly acting treatment option for patients living with IgAN.

Activation of Complement Factor C3/C3b Deposition on the Surface of Endothelial Cells by Histamine As one of the Causes of Endothelium Damage in COVID-19

Damage of the endothelium as a result of activation of the complement system is one of the causes of thrombotic complications in COVID-19. Factor C3 plays a key role in this process. The attachment of its proteolytic product C3b to the cells initiates the formation of the membrane attack complex C5b-9, which forms a pore in the plasma membrane and cell death. Here, we investigated how histamine, secreted in the body by leukocytes and mast cells, can affect the binding of C3b to human umbilical vein endothelial cells (HUVEC). To visualize it, FITS-conjugated antibodies against the C3c were used. These antibodies bind to intact C3 and to C3b but not to C3a. We have shown that when cultured HUVECs are incubated with human blood plasma, factor C3/C3b accumulates in the form of rounded and diffuse foci on the surface of the endothelial cell monolayer. Pre-activation of HUVEC by histamine increases the number of С3/C3b attachment sites. These data suggest that histamine may enhance endothelial damage during complement hyperactivation in COVID-19 and in endotheliopathies caused by other diseases.

Validation of dot blot immunoassay for measurement of complement opsonization of nanoparticles

Complement plays a critical role in the immune response toward nanomaterials. The complement attack on a foreign surface results in the deposition of C3, assembly of C3 convertases, the release of anaphylatoxins C3a and C5a, and finally, the formation of membrane attack complex C5b-9. Various technologies can measure complement activation markers in the fluid phase, but measurements of surface C3 deposition are less common. Previously, we developed an ultracentrifugation-based dot blot immunoassay (DBI) to measure the deposition of C3 and other protein corona components on nanoparticles. Here, we validate the repeatability of the DBI and its correlation with pathway-specific and common fluid phase markers. Moreover, we discuss the advantages of DBI, such as cost-effectiveness and versatility, while addressing potential limitations. This study provides insights into complement activation at the nanosurface level, offering a valuable tool for nanomedicine researchers in the field.

Increased Serum Terminal Complements Complex Levels in Attention Deficit Hyperactivity Disorder Children.

Attention deficit hyperactivity disorder (ADHD) is a widespread neuropsychiatric disorder in both children and adolescents, which is associated with social isolation and poor academic performance. Complement proteins are regarded as a major player in inflammation and disease development for several neuropsychiatric diseases such as schizophrenia and bipolar diseases. As clarified by previous data, increased levels of complement molecules and other immunological markers as cytokines were demonstrated in these disorders. Limited studies have investigated complement proteins particularly terminal complement complex or membrane attack complex (C5b-9) among ADHD patients. The present research aims to elucidate the association between C5b-9 complex protein and ADHD. This is a cross-sectional study. Sera were collected from Al-Hussain Teaching Medical City in Holy Karbala, Iraq, during 2019-2020. Sera were tested for C5-b9 using commercial kits by enzyme-linked immunosorbent assay (ELISA). In 90 participants included in the study, a significant increment in C5b-9 levels among ADHD patients (P=0.019) was observed. Patients with positive C5b-9 levels had a 2.76 times higher risk of developing ADHD than control subjects. The diagnostic utility for C5b-9 was statistically significant with 71.11% sensitivity, 55.6% specificity, and a high negative predictive value (97.3%). The study concluded elevation of the C5b-9 terminal complements complex levels in ADHD patients, which could point to the association of complement proteins as inflammatory markers with the ADHD disease process.

“Eculizumab First” in the Management of Posttransplant Thrombotic Microangiopathy

IntroductionPost-transplant thrombotic microangiopathy (PT-TMA) is an uncommon event that characterizes approximately 3-14% of kidney transplants (KT), and that is associated with a higher risk of delayed graft function and graft loss. PT-TMA occurs more frequently within the first three months after transplant and can be a manifestation of de novo disease or the recurrence of previous atypical hemolytic uremic syndrome (aHUS). Abnormalities in complement regulation genes could explain the increased susceptibility of some patients to PT-TMA. Eculizumab is a humanized monoclonal antibody that inhibits the formation of the membrane attack complex C5b-9. The aim of this study is to evaluate the efficacy of eculizumab as treatment for PT-TMA. MethodsWe retrospectively analyzed clinical records of 45 KT patients who received eculizumab immediately after the clinical diagnosis of PT-TMA. ResultsKidney biopsy was performed in 91.1% of patients and complement genetic study in 64.4%. 85.4% of kidney biopsies showed signs of TMA; genetic analysis revealed one pathogenetic variant, two variants of uncertain significance, one likely benign variant, 8 risk polymorphisms, 27 risk haplotypes.After two weeks from the treatment starting, hemoglobin and platelets significantly increased. A remarkable improvement in kidney function was also observed. After six months, 28.8% of patients had a complete renal recovery while 44.4% had a partial recovery. ConclusionThis is, to our knowledge, the largest series of KT patients with PT-TMA treated with eculizumab. These data suggest that eculizumab is associated with a normalization of hemolysis indices and an important and progressive improvement of graft function.

Preclinical efficacy of a cell division protein candidate gonococcal vaccine identified by artificial intelligence.

Vaccines to curb the global spread of multidrug-resistant gonorrhea are urgently needed. Here, 26 vaccine candidates identified by an artificial intelligence-driven platform (Efficacy Discriminative Educated Network[EDEN]) were screened for efficacy in the mouse vaginal colonization model. Complement-dependent bactericidal activity of antisera and the EDEN protective scores both correlated positively with the reduction in overall bacterial colonization burden. NGO1549 (FtsN) and NGO0265, both involved in cell division, displayed the best activity and were selected for further development. Both antigens, when fused to create a chimeric protein, elicited bactericidal antibodies against a wide array of gonococcal isolates and significantly attenuated the duration and burden of gonococcal colonization of mouse vaginas. Protection was abrogated in mice that lacked complement C9, the last step in the formation of the membrane attack complex pore, suggesting complement-dependent bactericidal activity as a mechanistic correlate of protection of the vaccine. FtsN and NGO0265 represent promising vaccine candidates against gonorrhea.

Multiple sclerosis plasma IgG aggregates induce complement-dependent neuronal apoptosis

Grey matter pathology is central to the progression of multiple sclerosis (MS). We discovered that MS plasma immunoglobulin G (IgG) antibodies, mainly IgG1, form large aggregates (>100 nm) which are retained in the flow-through after binding to Protein A. Utilizing an annexin V live-cell apoptosis detection assay, we demonstrated six times higher levels of neuronal apoptosis induced by MS plasma IgG aggregates (n = 190, from two cohorts) compared to other neurological disorders (n = 116) and healthy donors (n = 44). MS IgG aggregate-mediated, complement-dependent neuronal apoptosis was evaluated in multiple model systems including primary human neurons, primary human astrocytes, neuroblastoma SH-SY5Y cells, and newborn mouse brain slices. Immunocytochemistry revealed the co-deposition of IgG, early and late complement activation products (C1q, C3b, and membrane attack complex C5b9), as well as active caspase 3 in treated neuronal cells. Furthermore, we found that MS plasma cytotoxic antibodies are not present in Protein G flow-through, nor in the paired plasma. The neuronal apoptosis can be inhibited by IgG depletion, disruption of IgG aggregates, pan-caspase inhibitor, and is completely abolished by digestion with IgG-cleaving enzyme IdeS. Transmission electron microscopy and nanoparticle tracking analysis revealed the sizes of MS IgG aggregates are greater than 100 nm. Our data support the pathological role of MS IgG antibodies and corroborate their connection to complement activation and axonal damage, suggesting that apoptosis may be a mechanism of neurodegeneration in MS.

The neoepitope of the complement C5b-9 Membrane Attack Complex is formed by proximity of adjacent ancillary regions of C9

The Membrane Attack Complex (MAC) is responsible for forming large β-barrel channels in the membranes of pathogens, such as gram-negative bacteria. Off-target MAC assembly on endogenous tissue is associated with inflammatory diseases and cancer. Accordingly, a human C5b-9 specific antibody, aE11, has been developed that detects a neoepitope exposed in C9 when it is incorporated into the C5b-9 complex, but not present in the plasma native C9. For nearly four decades aE11 has been routinely used to study complement, MAC-related inflammation, and pathophysiology. However, the identity of C9 neoepitope remains unknown. Here, we determined the cryo-EM structure of aE11 in complex with polyC9 at 3.2 Å resolution. The aE11 binding site is formed by two separate surfaces of the oligomeric C9 periphery and is therefore a discontinuous quaternary epitope. These surfaces are contributed by portions of the adjacent TSP1, LDLRA, and MACPF domains of two neighbouring C9 protomers. By substituting key antibody interacting residues to the murine orthologue, we validated the unusual binding modality of aE11. Furthermore, aE11 can recognise a partial epitope in purified monomeric C9 in vitro, albeit weakly. Taken together, our results reveal the structural basis for MAC recognition by aE11.

Pegcetacoplan Corrects Hyperbilirubinemia in Both Untreated Patients with Paroxysmal Nocturnal Hemoglobinuria (PNH) and in Patients with PNH Who Previously Received Eculizumab

Background Paroxysmal nocturnal hemoglobinuria (PNH) is a rare, acquired disease characterized by complement-mediated hemolysis and thrombosis. It results from loss of the glycosylphosphatidylinositol-linked complement-inhibitor proteins CD55 and CD59 that inhibit early and late complement proteins, respectively, on red blood cell (RBC) membranes. In untreated patients, formation of the C5b-9 membrane attack complex results in chronic intravascular hemolysis (IVH) and acute hemolytic attacks. Treatment with the C5 inhibitors eculizumab (ECU) and ravulizumab inhibits IVH and unmasks extravascular hemolysis (EVH), which is due to C3b-mediated opsonization of RBCs and their clearance by the reticuloendothelial system. This can result in unconjugated (indirect) hyperbilirubinemia, leading to jaundice and scleral icterus when plasma bilirubin levels reach 2.0-3.0 mg/dL. As in other conditions with EVH (eg, sickle cell disease), bilirubin gallstones have been reported in patients with PNH treated with C5 inhibitors. Pegcetacoplan (PEG), a C3 inhibitor that controls IVH and EVH, is approved in the US for the treatment of adults with PNH. We hypothesized that PEG could control hyperbilirubinemia in PNH patients, reducing jaundice and the risk of gallstone formation. Methods This post hoc analysis evaluated 16- and 26-week data from 2 Phase 3 randomized controlled trials. PEGASUS (NCT03500549) evaluated patients with hemoglobin <10.5 g/dL despite stable ECU treatment for 48 weeks; patients received PEG or ECU for 16 weeks. PRINCE (NCT04085601) evaluated PEG therapy for 26 weeks in patients who were untreated with complement inhibitors. Mean and median total and indirect bilirubin values were calculated at baseline and at the end of the treatment period for patients treated with PEG and ECU in PEGASUS and with PEG in PRINCE. Total bilirubin plasma levels were categorized as normal (<1.1 mg/dL), intermediate (1.1-3.0 mg/dL), and high (>3.0 mg/dL). The main endpoint was the distribution of bilirubin categories at baseline compared with the end of the treatment period and probability of normalization as analyzed by Bowker's test of symmetry. Patients were excluded from the analysis if they did not have both a baseline and end of treatment bilirubin value for analysis. Results In PEGASUS, 74 of 80 patients (PEG, n=36; ECU, n=38) had evaluable bilirubin data; 30 of 35 PEG-treated patients were evaluable from PRINCE. At baseline, 85% (n=63) of patients in PEGASUS had abnormal bilirubin values and 26% (n=20) had high bilirubin values. In PRINCE at baseline, 87% had abnormal bilirubin values, and 23% had high bilirubin values. The hyperbilirubinemia was primarily of the indirect form (TABLE). At 16 weeks, 72% of patients treated with PEG in PEGASUS had normal bilirubin values compared with 16% of patients treated with ECU. In PRINCE, 83% of patients treated with PEG had normal bilirubin at 26 weeks; none had high bilirubin values. The shifts toward normalization in bilirubin categorization (FIGURE) were statistically significant (p<0.0001) for patients on PEG therapy in both PEGASUS and PRINCE. Continued treatment with ECU was not associated with a significant change in bilirubin (p=0.39). In PEGASUS, the normalization rate was 71% (n=22/31; exact 95% CI: 52%, 86%) for PEG and 9% (n=3/32; exact CI: 2%, 25%) for ECU. In PRINCE, the normalization rate was 81% (n=21/26 patients; exact CI: 61%, 93%) after PEG treatment. Conclusions This post hoc analysis found a high prevalence of abnormal bilirubin values at baseline in patients with PNH who were treated with a C5 inhibitor or were untreated. Treatment with PEG significantly improved bilirubin levels, with the majority of patients shifting to normal values. Conversely, bilirubin levels did not improve with continued ECU treatment. These findings suggest that long-term treatment with PEG reduces the signs and symptoms of hyperbilirubinemia, improving jaundice, and decreasing the risk of bilirubin stones and associated complications (eg, biliary sepsis and pancreatitis) in patients with PNH. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

GLOMERULAR MICROANGIOPATHY WITH MARKED SYSTEMIC THROMBOTIC MICROANGIOPATHY SHORTLY AFTER BORTEZOMIB IN A NEWLY DIAGNOSED POEMS SYNDROME PATIENT

AbstractThe dipeptide boronic acid analogue bortezomib as a potent and selective inhibitor of the proteasome is used for the treatment of plasma cell dyscrasias such as multiple myeloma (MM). Bortezomib may induce glomerular microangiopathy (GMA) with or without systemic thrombotic microangiopathy (TMA) in which vascular endothelial growth factor-nuclear factor (VEGF) -κ B pathway could be involved. MM itself can cause TMA but primarily at presentation. Case reportWe present a case with GMA associated with clinical features supporting systemic TMA shortly after bortezomib. Case: A 54-year-old woman has been diagnosed as having POEMS syndrome. She had symmetric mild degree of peripheral neuropathy, scleroatrophic skin lesions, Raynaud's phenomenon, and retinopathy. IgG kappa type paraproteinemia with a monotypic increase of plasma cells and increased pulmonary artery pressure contributed to the diagnosis. Bortezomib based treatment was started. MethodologyAt the 20th day she developed severe dyspnea. Bilateral pleural effusion and acute kidney failure with thrombocytopenia and microangiopathic hemolytic anemia were documented. Urgent steroid and plasmapheresis were started. ADAMTS13 level proved to be within normal and plasmapheresis did not contribute to improvement. She commenced on hemodialysis and kidney biopsy was decided. Light microscopy findings revealed glomerular capillary thrombus, basement membrane thickening and segmental Resultsduplication. Hyperplastic arteriolar changes were present. No immune deposits were detected by immunofluorescence microscopy. Biopsy findings were diagnostic for thrombotic microangiopathy. The clinical picture deteriorated as sleepiness and confusion. Cranial imaging and cerebrospinal fluid analysis showed no abnormality. Eculizumab with off-label approval contributed to stabilization but no improvement. ConclusionConclusion: Proteasome inhibitors associated with TMA may be life-threatening along with organ dysfunction due to microangiopathy-related ischemia. Membrane attack complex (C5b-9) deposition was found on endothelial cells in culture exposed to plasma from patients during the acute phase of the disease which may point to complement blockade benefit.

Saponins from Panax notoginseng ameliorate steroid resistance in lupus nephritis through regulating lymphocyte-derived exosomes in mice.

Lupus nephritis (LN) is the most common and severe type of organ damage and an important primary disease in end-stage renal failure in patients with systemic lupus erythematosus (SLE). Clinical guidelines recommend steroid treatment, but steroid resistance has become a major factor leading to treatment failure and affecting prognosis. Our previous study demonstrated that Saponins from Panax Notoginseng (Panax ginseng saponins, PNS) could reverse steroid resistance of lymphocytes by downregulating P-glycoprotein (P-gp) expression and provide renal protection in LN mice, but the mechanism by which lymphocytes transmit these related messages to renal lamina propria cells is not clear. Therefore, we further elucidated this mechanism through holistic experiments. In this study, low-dose methylprednisolone (0.8 mg/kg/day, MP) was used to induce a steroid-resistant lupus nephritis (SR-LN) mouse model in weeks one to four, and a therapeutic steroid dosage (MP 12 mg/kg/day) or a combined PNS (PNS 100 mg/kg/day) treatment was administered from week five to eight. Lymphocyte-derived exosomes (Lyme-Exos) were isolated from the spleens of mice and injected into untreated homozygous LN mice for 14 days via the tail vein. At the end of the experiment, the efficacy and mechanism of action of different groups of Lyme-Exos on LN mice were observed. The results revealed that exogenously injected Lyme-Exos were effectively taken up by the kidney and affected the progression of kidney disease. Steroid-resistant lymphocyte-derived exosomes intervented with PNS significantly downregulated the levels of silent information regulator-related enzyme 1 (Sirt1), multidrug resistance gene 1 (MDR1), and P-gp in the renal cortex and glomerular endothelial cells (GECs); reduced serum autoantibody [antinuclear antibody (ANA) and anti-double-stranded DNA (dsDNA)] levels and inflammatory markers (WBC, PCR, and PCT); improved renal function; and attenuated urinary microalbumin excretion. Additionally, renal histopathological damage (HE staining) and fibrosis (Masson staining) were improved, and immune complex (IgG) deposition and membrane attack complex (C5b-9) production were significantly reduced; the gene levels of inflammatory factors (INF-γ, MCP-1, IL-8, IL-17, vWF, VCAM-1, IL-1β, IL-6, PTX3) in the renal cortex were downregulated. Taken together, this study showed that PNS may alleviate steroid resistance in GEC by interfering with steroid-resistant Lyme-Exos to ameliorate LN progression, which will likely provide insights into developing a new LN treatment.

Immature Platelet Fraction and Thrombin Generation: Preeclampsia Biomarkers.

Preeclampsia, a human pregnancy syndrome, is characterized by elevated blood pressure and proteinuria after the 20th week of gestation. Its etiology remains unknown, and its pathophysiological mechanisms are related to placental hypoperfusion, endothelial dysfunction, inflammation, and coagulation cascade activation. Recently, the role of the complement system has been considered. This syndrome is one of the main causes of maternal and fetal mortality and morbidity. This article discusses the hypothesis of preeclampsia being triggered by the occurrence of inadequate implantation of the syncytiotrophoblast, associated with bleeding during the first stage of pregnancy and with augmented thrombin generation. Thrombin activates platelets, increasing the release of antiangiogenic factors and activating the complement system, inducing the membrane attack complex (C5b9). Immature platelet fraction and thrombin generation may be possible blood biomarkers to help the early diagnosis of preeclampsia.

Membrane Attack Complex C5b-9 Promotes Renal Tubular Epithelial Cell Pyroptosis in Trichloroethylene-Sensitized Mice.

Trichloroethylene (TCE), a commonly used organic solvent, is known to cause trichloroethylene hypersensitivity syndrome (THS), also called occupational medicamentosa–like dermatitis due to TCE (OMDT) in China. OMDT patients presented with severe inflammatory kidney damage, and we have previously shown that the renal damage is related to the terminal complement complex C5b-9. Here, we sought to determine whether C5b-9 participated in TCE-induced immune kidney injury by promoting pyroptosis, a new form of programed cell death linked to inflammatory response, with underlying molecular mechanisms involving the NLRP3 inflammasome. A BALB/c mouse-based model of OMDT was established by dermal TCE sensitization in the presence or absence of C5b-9 inhibitor (sCD59-Cys, 25μg/mouse) and NLRP3 antagonist (MCC950, 10 mg/kg). Kidney histopathology, renal function, expression of inflammatory mediators and the pyroptosis executive protein gasdermin D (GSDMD), and the activation of pyroptosis canonical NLRP3/caspase-1 pathway were examined in the mouse model. Renal tubular damage was observed in TCE-sensitized mice. GSDMD was mainly expressed on renal tubular epithelial cells (RTECs). The caspase-1–dependent canonical pathway of pyroptosis was activated in TCE-induced renal damage. Pharmacological inhibition of C5b-9 could restrain the caspase-1–dependent canonical pathway and rescued the renal tubular damage. Taken together, our results demonstrated that complement C5b-9 plays a central role in TCE-induced immune kidney damage, and the underlying mechanisms involve NLRP3-mediated pyroptosis.

MO241: Nets and Terminal Complement Pathway as Potential Biomarkers for Complement Overactivation Assessment in Anca-Associated Vasculitis

Abstract BACKGROUND AND AIMS Clinical, in vitro, and animal model-derived evidence has demonstrated a critical involvement of the alternative complement pathway (aCP) in the pathogenesis of ANCA-associated vasculitis (AAV). In this regard, neutrophil extracellular traps (NETs) have been suggested to be a key element between ANCA-induced neutrophil activation and aCP [1]. However, the role of the terminal complement pathway (tCP) is less well studied [2]. METHOD A prospective, observational, multicenter study analyzing first episodes and relapses of patients with AAV, with a minimum follow-up of 6 months, was performed. Blood samples were collected at diagnosis (AAV-t1) and at remission (AAV-t2). Control population consisted of age and sex-matched individuals. Complement activation was assessed by analyzing the complement membrane attack complex (C5b-9) deposition on cultured endothelial cells (HMEC-1), by immunofluorescence, after exposing them to activated plasma (a-plasma: obtained by mixing patient's citrated plasma with healthy subjects’ sera pool, 1:1). C5b-9 deposits induced by patients’ a-plasma were calculated as percentage of labeled area with respect to the total area analyzed. Results from patient and control samples were expressed as fold increase (mean ± SEM) versus those obtained with the pool of a-plasma from healthy subjects. Plasma levels of tCP and aCP soluble factors, such as sC5b-9 and sFBb (respectively), were also measured (mean ± SEM). Circulating NETs were indirectly measured by quantifying circulant dsDNA plasmatic concentration (mean ± SEM) as a NET surrogate. RESULTS The present results were obtained with samples from 13 AAV-MPO patients who achieved complete remission (38% men, age 63 ± 14 years) and 10 controls (45% men, age 66 ± 6 years). At AAV-t1, there was a statistically significant increase (P &amp;lt; .05) of C5b-9 deposition on HMEC-1 in response to patients’ a-plasma (fold increase of 5.3 ± 1.3) compared with control samples (fold increase of 1.2 ± 0.2). Samples obtained at AAV-t2 induced less C5b-9 deposition than at AAV-t1 (fold increase of 0.9 ± 0.2; P &amp;lt; .05), with values similar to controls. Regarding soluble factors, levels of both sC5b-9 and sFBb were significantly increased in AAV-t1 (1882 ± 418 ng/mL and 3.2 ± 0.4 µg/mL, respectively; P &amp;lt; .05) versus AAV-t2 (852 ± 104 ng/mL and 1.9 ± 0.2 µg/mL, respectively; P &amp;lt; .05). At AAV-t2, levels were similar to controls (708 ± 42 ng/mL for sC5b-9 and 2.4 ± 0.2 µg/mL for sFBb). Circulating NETs were also increased in AAV-t1 (22.2 ± 3.5 µg/mL) compared with both AAV-t2 and controls (13.6 ± 1.1 µg/mL and 13.7 ± 0.3 µg/mL, respectively; P &amp;lt; .05). Moreover, NETs in AAV-t1 presented a significant correlation with sFBb levels, both in AAV-t1 (r = 0.709; P &amp;lt; .05) and in AAV-t2 (r = 0.585; P &amp;lt; .05). CONCLUSION There is a relationship between NETs, AAV activity and sFBb, which supports the role of NETs and cAP in AAV pathogenesis. Moreover, differences in C5b-9 deposition between the two stages of the disease suggest that tCP may be dysregulated in AAV. Further characterization of this dysregulation may lead to new diagnostic or disease activity biomarkers, as well as new therapeutic options for the management of patients with AAV.

C5b-9 Membrane Attack Complex Formation andExtracellular Vesicle Shedding in Barrett's Esophagus and Esophageal Adenocarcinoma.

The early complement components have emerged as mediators of pro-oncogenic inflammation, classically inferred to cause terminal complement activation, but there are limited data on the activity of terminal complement in cancer. We previously reported elevated serum and tissue C9, the terminal complement component, in esophageal adenocarcinoma (EAC) compared to the precursor condition Barrett’s Esophagus (BE) and healthy controls. Here, we investigate the level and cellular fates of the terminal complement complex C5b-9, also known as the membrane attack complex. Punctate C5b-9 staining and diffuse C9 staining was detected in BE and EAC by multiplex immunohistofluorescence without corresponding increase of C9 mRNA transcript. Increased C9 and C5b-9 staining were observed in the sequence normal squamous epithelium, BE, low- and high-grade dysplasia, EAC. C5b-9 positive esophageal cells were morphologically intact, indicative of sublytic or complement-evasion mechanisms. To investigate this at a cellular level, we exposed non-dysplastic BE (BAR-T and CP-A), high-grade dysplastic BE (CP-B and CP-D) and EAC (FLO-1 and OE-33) cell lines to the same sublytic dose of immunopurified human C9 (3 µg/ml) in the presence of C9-depleted human serum. Cellular C5b-9 was visualized by immunofluorescence confocal microscopy. Shed C5b-9 in the form of extracellular vesicles (EV) was measured in collected conditioned medium using recently described microfluidic immunoassay with capture by a mixture of three tetraspanin antibodies (CD9/CD63/CD81) and detection by surface-enhanced Raman scattering (SERS) after EV labelling with C5b-9 or C9 antibody conjugated SERS nanotags. Following C9 exposure, all examined cell lines formed C5b-9, internalized C5b-9, and shed C5b-9+ and C9+ EVs, albeit at varying levels despite receiving the same C9 dose. In conclusion, these results confirm increased esophageal C5b-9 formation during EAC development and demonstrate capability and heterogeneity in C5b-9 formation and shedding in BE and EAC cell lines following sublytic C9 exposure. Future work may explore the molecular mechanisms and pathogenic implications of the shed C5b-9+ EV.

Diversity of complement activation in different thyroid diseases

ObjectiveTo investigate complement components expression in both thyroid tissues and serum from patients with Hashimoto's thyroiditis (HT), Graves' disease (GD), and papillary thyroid cancer (PTC). MethodsC1q, mannose binding lectin (MBL), Bb, C4d, C3d and membrane attack complex (MAC) (C5b-9) deposition and complement regulate proteins (CD46, CD55 and CD59) expression in thyroid tissues from HT, GD, PTC, and control groups were examined by IHC. C1q, MBL, Bb, C4d, C3a, and soluble C5b-9 (sC5b-9) serum levels in the HT, GD, PTC, and healthy donor (HD) groups were measured by ELISAs. ResultsMAC deposition was detected in thyroid tissues in the HT, GD and PTC groups, but not the control group. MBL, Bb, C4d, C3d and MAC staining intensities in thyroid tissues were significantly higher in the HT and PTC groups than in the control group (all P < 0.05). The C1q level was higher in HT tissues than in control tissues (both P < 0.05). No complement component had a significant difference in staining intensities between the GD and control groups. CD55 and CD59 expression levels in thyroid tissues were higher in the PTC group than in the HT, GD and control groups (all P < 0.05). Similarly, CD46 levels were higher in HT tissues than in control tissues. Bb, C4d, C3a and sC5b-9 serum levels were significantly increased in HT, GD and PTC patients compared with HDs (all P < 0.05). ConclusionComplement is overactivated in HT and PTC, but not in GD. All the three pathways are activated in HT, and the MBL and alternative complement pathways are activated in PTC. These distinct complement activation profiles may participate in HT, GD and PTC pathogenesis.

Effect of Nephritis Rehabilitation Tablets combined with tacrolimus in treatment of idiopathic membranous nephropathy.

BACKGROUNDIdiopathic membranous nephropathy (IMN) has a high incidence in the middle-aged and elderly population, and poses a great threat to the physical and mental health and quality of life of patients. Nephritis Rehabilitation Tablets have many potential effects, such as clearing residual toxins, tumefying the kidney and spleen, replenishing qi, and nourishing yin, and have played an important role in the treatment of a variety of kidney diseases.AIMTo investigate the efficacy and safety of Nephritis Rehabilitation Tablets combined with tacrolimus in the treatment of IMN.METHODSEighty-four patients with IMN recruited from January 2017 to September 2020 were randomly divided into a study group (n = 42) and a control group (n = 42). On the basis of routine symptomatic treatment, both groups were treated with tacrolimus, and the study group was additionally treated with Nephritis Rehabilitation Tablets. Both groups were treated for 12 wk. The therapeutic effect, the levels of renal function indexes [serum creatinine (Scr), serum albumin, and 24-h urinary protein], urinary immunoglobulin (IgG4), membrane attack complex (C5b-9), and the incidence of adverse reactions were measured before and after 12 wk of treatment.RESULTSThe total effective rate in the study group was significantly higher than that of the control group. Before treatment, there was no significant difference in Scr, serum albumin, or 24 h urinary protein between the two groups. After 12 wk of treatment, the levels of Scr and 24-h urinary protein in both groups were significantly lower and serum albumin was significantly higher than those before treatment (P < 0.05), and the levels of Scr and 24-h urinary protein were significantly lower (P = 0.003 and 0.000, respectively), and the level of serum albumin was significantly higher (P = 0.00) in the study group than in the control group. Before treatment, there was no significant difference in urinary IgG4 and C5b-9 levels between the study group and the control group (P = 0.336 and 0.438, respectively). After 12 wk of treatment, the levels of urinary IgG4 and C5b-9 in the two groups were lower than those before treatment, and the levels of urinary IgG4 and C5b-9 in the study group were significantly lower than those in the control group (P = 0.000). There was no significant difference in the incidence of adverse reactions between the two groups (P = 0.710).CONCLUSIONBased on routine intervention, Nephritis Rehabilitation Tablets combined with tacrolimus in the treatment of IMN can effectively improve the renal function of patients and downregulate the expression of urinary IgG4 and C5b-9. In addition, they can improve the overall therapeutic effect while not increasing the risk of adverse reactions.

Complement dysregulation is associated with severe COVID-19 illness.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) may manifest as thrombosis, stroke, renal failure, myocardial infarction, and thrombocytopenia, reminiscent of other complement- mediated diseases. Multiple clinical and preclinical studies have implicated complement in the pathogenesis of COVID-19 illness. We previously found that the SARS-CoV-2 spike protein activates the alternative pathway of complement (APC) in vitro through interfering with the function of complement factor H, a key negative regulator of APC. Here, we demonstrated that serum from 58 COVID-19 patients (32 patients with minimal oxygen requirement, 7 on high flow oxygen, 17 requiring mechanical ventilation and 2 deaths) can induce complementmediated cell death in a functional assay (the modified Ham test) and increase membrane attack complex (C5b-9) deposition on the cell surface. A positive modified Ham assay (>20% cell-killing) was present in 41.2% COVID-19 patients requiring intubation (n=7/17) and only 6.3% in COVID-19 patients requiring minimal oxygen support (n=2/32). C5 and factor D inhibition effectively mitigated the complement amplification induced by COVID-19 patient serum. Increased serum factor Bb level was associated with disease severity in COVID-19 patients, suggesting that APC dysregulation plays an important role. Moreover, SARS-CoV-2 spike proteins directly block complement factor H from binding to heparin, which may lead to complement dysregulation on the cell surface. Taken together, our data suggest that complement dysregulation contributes to the pathogenesis of COVID-19 and may be a marker of disease severity.

Response Rates, Non-Relapse Mortality, and Financial Implications of Transplant-Associated Thrombotic Microangiopathy Treated with Eculizumab

Background: Hematopoietic cell transplantation associated thrombotic microangiopathy (TA-TMA) shares common features with atypical hemolytic uremic syndrome (aHUS) and thrombotic thrombocytopenic purpura. TA-TMA affects multiple organ systems resulting in multi-organ failure and non-relapse mortality (NRM). Recently, complement activation has been associated with TA-TMA leading to utilization of complement blockade in TMA. Eculizumab is a humanized monoclonal antibody that binds to C5 and prevents the formation of the membrane attack complex (C5b-9) or the terminal complement pathway. Eculizumab may be an effective treatment for pediatric TA-TMA patients; however, there are limited data regarding its use in adult TA-TMA patients. Furthermore, eculizumab is an expensive medication and there are no published data regarding the financial implications of TA-TMA treated with eculizumab. The purpose of this study was to examine the response rates, NRM, and financial implications of TA-TMA treated with eculizumab.Methods: We performed a retrospective cohort study of patients with TA-TMA who received eculizumab at our institution between January 1, 2012, and April 9, 2017. TA-TMA was defined according to the BMT-CTN criteria with evidence of hemolysis, elevated LDH, renal dysfunction and negative Coombs' test. Serum complement biomarkers (C5b-9) were used to support the diagnosis when platelet or red blood cell transfusion dependence occurred with organ dysfunction or tissue evidence of microangiopathy. Patients were vaccinated against Neisseria meningitides and received appropriate antibiotic prophylaxis. Eculizumab therapy was initiated at the Food and Drug Administration-approved dosing schedule for aHUS: 900 mg intravenously weekly for the first 4 weeks followed by 1200 mg intravenously every other week beginning on week 5 for a total of 8 weeks, with some patients requiring longer than an 8 week course. The primary outcome was complete response to eculizumab therapy and responders were defined by achievement of transfusion independence and improvement in creatinine. Secondary outcomes included non-relapse mortality (NRM), inpatient cost, number of comorbidities, number of infections, number of hospitalizations, and hospital length of stay (LOS). Comorbidities were defined as any documented medical problem other than the reason for transplant and TA-TMA.Results: Twenty patients were included in the study (Table 1). The median (range) time from transplant to diagnosis of TA-TMA was 139 (14-388) days. The median (range) time from diagnosis of TA-TMA to first dose of eculizumab was 2 (0-272) days. The complete response rate was 55%. The median (range) number of doses of eculizumab was 6.5 (2-17) for responders compared to 3 (1-7) for non-responders including both inpatient and outpatient doses (p= 0.01). There were no significant differences between the number of hospitalizations for responders and non-responders. However, responders had shorter LOS and lower inpatient costs than non-responders. The median (range) LOS was 9 days (0-92) for responders compared to 61 days (26-94) for non-responders (p= 0.03). The median (range) total inpatient cost was $259,734 (0-1,670,070) for responders compared to $1,525,758 (385,092-1,948,190) for non-responders (p= 0.003). The itemized inpatient costs are shown in Figure 1. Patients who responded to eculizumab tended to be younger with less comorbidities. In fact, patients were 33% less likely to respond to eculizumab for every additional documented comorbidity at the time of TA-TMA diagnosis (HR=0.67 [95% CI 0.49-0.92]). The NRM was 1/11 (9%) for responders and 9/9 (100%) for non-responders (HR=0.07 [95% CI: 0.007-0.66]).Conclusions: TA-TMA is an important complication of HCT that is associated with high NRM and financial toxicity. Our data suggests that patients who respond to eculizumab have lower NRM, LOS, and financial costs than non-responders. There was a statistically non-significant trend in incidence of steroid-refractory GVHD in the non-responder group; additionally non-responders received significantly fewer doses than responders, which may explain partly the lower response rates compared to the 80% response rates in aHUS. Patient age and the number of comorbidities at the time of diagnosis of TA-TMA may be important predictive factors for response to eculizumab. [Display omitted] DisclosuresMims:Novartis: Honoraria. Hofmeister:Janssen: Research Funding; Karyopharm: Research Funding; Celgene: Research Funding; Thrassos: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Research Funding; Adaptive Biotechnologies: Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Research Funding; Takeda: Research Funding. Walker:Gilead: Research Funding. Vasu:Stemline Therapeutics: Research Funding; Boehringer-Ingelheim: Research Funding.