Terminal Complement Components Research Articles

Complement associated microvascular injury and thrombosis in the pathogenesis of severe COVID-19 infection: A report of five cases

Acute respiratory failure and a systemic coagulopathy are critical aspects of the morbidity and mortality characterizing infection with severe acute respiratory distress syndrome-associated coronavirus-2, the etiologic agent of Coronavirus disease 2019 (COVID-19). We examined skin and lung tissues from 5 patients with severe COVID-19 characterized by respiratory failure (n= 5) and purpuric skin rash (n = 3). COVID-19 pneumonitis was predominantly a pauci-inflammatory septal capillary injury with significant septal capillary mural and luminal fibrin deposition and permeation of the interalveolar septa by neutrophils. No viral cytopathic changes were observed and the diffuse alveolar damage (DAD) with hyaline membranes, inflammation, and type II pneumocyte hyperplasia, hallmarks of classic acute respiratory distress syndrome, were not prominent. These pulmonary findings were accompanied by significant deposits of terminal complement components C5b-9 (membrane attack complex), C4d, and mannose binding lectin (MBL)-associated serine protease (MASP)2, in the microvasculature, consistent with sustained, systemic activation of the complement pathways. The purpuric skin lesions similarly showed a pauci-inflammatory thrombogenic vasculopathy, with deposition of C5b-9 and C4d in both grossly involved and normally-appearing skin. In addition, there was co-localization of COVID-19 spike glycoproteins with C4d and C5b-9 in the interalveolar septa and the cutaneous microvasculature of 2 cases examined. In conclusion, at least a subset of sustained, severe COVID-19 may define a type of catastrophic microvascular injury syndrome mediated by activation of complement pathways and an associated procoagulant state. It provides a foundation for further exploration of the pathophysiologic importance of complement in COVID-19, and could suggest targets for specific intervention.

Characterization and expression analysis of the C8α and C9 terminal complement components from pufferfish (Takifugu rubripes)

C8α and C9 mediate the membrane attack complex formation and bacterial lysis and are important components in the complement system. The cDNA sequences of the C8α and C9 genes were cloned from Takifugu rubripes. The full-length cDNA of Tr-C8α was 1893 bp and included a 5′-UTR of 69 bp and 3′-UTR of 83 bp. The full-length cDNA of Tr-C9 was 2083 bp and included a 5′-UTR of 72 bp and 3′-UTR of 250 bp. The expression of Tr-C8α and Tr-C9 was detected in newly fertilized eggs of T. rubripes. The expression of these two genes was at a higher level in the liver than in other tissues tested. After lipopolysaccharide (LPS) challenge, the gene expression of Tr-C8α and Tr-C9 increased more significantly in the liver. With these combined results, we further understood how Tr-C8α and Tr-C9 function in the innate immunity of pufferfish. Our findings could deepen the understanding of immune regulation in pufferfish.

Consensus regarding diagnosis and management of atypical hemolytic uremic syndrome.

Thrombotic microangiopathy (TMA) is defined by specific clinical characteristics, including microangiopathic hemolytic anemia, thrombocytopenia, and pathologic evidence of endothelial cell damage, as well as the resulting ischemic end-organ injuries. A variety of clinical scenarios have features of TMA, including infection, pregnancy, malignancy, autoimmune disease, and medications. These overlapping manifestations hamper differential diagnosis of the underlying pathogenesis, despite recent advances in understanding the mechanisms of several types of TMA syndrome. Atypical hemolytic uremic syndrome (aHUS) is caused by a genetic or acquired defect in regulation of the alternative complement pathway. It is important to consider the possibility of aHUS in all patients who exhibit TMA with triggering conditions because of the incomplete genetic penetrance of aHUS. Therapeutic strategies for aHUS are based on functional restoration of the complement system. Eculizumab, a monoclonal antibody against the terminal complement component 5 inhibitor, yields good outcomes that include prevention of organ damage and premature death. However, there remain unresolved challenges in terms of treatment duration, cost, and infectious complications. A consensus regarding diagnosis and management of TMA syndrome would enhance understanding of the disease and enable treatment decision-making.

Molecular characterization and induced expression analysis of the terminal complement component C9 in rohu,Labeo rohita

The complement component 9 (C9) plays a significant role in the formation of membrane attack complex (MAC) on the targeted cell surface. The current study is dealt with molecular characterization of C9 gene from rohu, Labeo rohita, an important cultured carp species in India. An open reading frame (ORF) of 1998 bp was amplified by polymerase chain reaction (PCR) that encodes a polypeptide of 666 amino acids having a signal peptide of 19 amino acids and a mature peptide of 647 amino acids. The SMART domain architecture analysis revealed two thrombospondin type-1 domains (TSP1), a low-density lipoprotein receptor domain class A (LDLa), a membrane attack complex and perforin (MACPF) domain, and an epidermal growth factor (EGF)-like domain. Multiple sequence alignment and evolutionary analysis revealed a primitive C9 sequence of rohu with maximum similarity and clustering with common carp, grass carp and zebrafish. C9 was highly expressed in liver and constitutively expressed in wide array of tissues except in eye of rohu juveniles. It was expressed during early developmental days of rohu including in milt. A variable level of up-regulation in C9 expression was noticed upon poly I:C induction, Aeromonas hydrophila and Argulus siamensis infections in liver, spleen and gill tissues of rohu at different time points. A constitutive expression of C9 in different stages of rohu during the ontogeny and in response to pathogen exposures along with high degree of sequence homology with other fish species proved it as an important primitive immune molecule of the complement system lytic pathway.

Cell-Type-Specific Complement Expression in the Healthy and Diseased Retina

SUMMARYComplement dysregulation is a feature of many retinal diseases, yet mechanistic understanding at the cellular level is limited. Given this knowledge gap about which retinal cells express complement, we performed single-cell RNA sequencing on ~92,000 mouse retinal cells and validated our results in five major purified retinal cell types. We found evidence for a distributed cell-type-specific complement expression across 11 cell types. Notably, Müller cells are the major contributor of complement activators c1s, c3, c4, and cfb. Retinal pigment epithelium (RPE) mainly expresses cfh and the terminal complement components, whereas cfi and cfp transcripts are most abundant in neurons. Aging enhances c1s, cfb, cfp, and cfi expression, while cfh expression decreases. Transient retinal ischemia increases complement expression in microglia, Müller cells, and RPE. In summary, we report a unique complement expression signature for murine retinal cell types suggesting a well-orchestrated regulation of local complement expression in the retinal microenvironment.

Sublytic Terminal Complement Components Induce Eryptosis in Autoimmune Haemolytic Anaemia Related to IgM Autoantibodies.

Eryptosis, the suicidal death of red blood cells (RBCs), is characterized by phosphatidylserine (PS) exposure at the cell surface. It can be catalysed by a variety of abnormal conditions and diseases. Until now, the many questions surrounding the physiology and pathophysiology of eryptosis have not been sufficiently answered. Recently, we demonstrated IgM and IgA autoantibodies (aab) to induce PS exposure on circulating RBCs of patients with autoimmune haemolytic anaemia (AIHA). However, it remained unclear how these aab lead to eryptosis. Serum and plasma samples from patients with clinically relevant AIHA of cold type were used to induce eryptosis in O RBCs. Serum containing fresh complement from healthy donors, antibodies to complement component, and complement factor depleted sera were added to examine the influence of the complement on PS-exposure. RBC bound annexin V PE were analysed by flow cytometry. Eryptosis related to IgM aab was found to be dependent on complement activation and could be effectively inhibited by EDTA, serum heat inactivation and anti-C5. PS exposure increased with sequential activation of the sublytic terminal complement components C5b6, C5b-7 and was most significant at the C5b-8 stage. A decrease was observed following the formation of the lytic membrane attack complex C5b-9, either because of lysis of eryptotic RBCs or because of inhibition of eryptosis by C9. Our findings reflect new aspects on RBC destruction in AIHA as well the impact of the terminal complement complexes on the RBC membrane. The striking differences to nucleated cell apoptosis may even have physiological meaning of RBC acting as a buffer of the complement system.

Microparticles Expressing Myeloperoxidase and Complement C3a and C5a as Markers of Renal Involvement in Antineutrophil Cytoplasmic Antibody-associated Vasculitis.

To investigate expression of terminal complement components C3a and C5a on circulating myeloperoxidase (MPO)-positive microparticles (MPO+MP) in relation to disease activity and renal involvement in patients with antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). Forty-six clinically well-characterized patients with AAV and 23 age- and sex-matched healthy controls were included. The concentration of MPO+MP expressing C3a and C5a was analyzed from citrate plasma by flow cytometry. Serum levels of C3a and C5a were determined using commercial ELISA. The assessment of vasculitis disease activity was performed using the Birmingham Vasculitis Activity Score (BVAS). Among patients, 23 had active disease with BVAS ≥ 2 and 14 patients had active renal flares. AAV patients had significantly increased expression of C3a and C5a on MPO+MP compared to controls (both p < 0.0001). When the group of patients with active AAV was divided according to the presence of renal activity, the concentration of MPO+MP expressing C3a and C5a was significantly higher in patients with renal involvement compared to patients with nonrenal disease and controls (p < 0.05 and p < 0.01, respectively). The serum levels of C3a were significantly decreased (p < 0.01) in the renal subgroup, while there were no changes in serum levels of C5a comparing the renal and nonrenal groups. There was significant correlation between the disease activity measured by BVAS and the levels of C3a and C5a expressed on MPO+MP. Determination of C3a and C5a on MPO+MP might be considered as a novel biomarker of renal involvement in patients with AAV and may be of importance in the pathogenetic process.

Investigational RNAi Therapeutic Targeting C5 Is Efficacious in Pre-clinical Models of Myasthenia Gravis

Complement-mediated damage to the neuromuscular junction (NMJ) is a key mechanism of pathology in myasthenia gravis (MG), and therapeutics inhibiting complement have shown evidence of efficacy in the treatment of MG. In this study, we describe the development of a subcutaneously administered N-acetylgalactosamine (GalNAc)-conjugated small interfering RNA (siRNA) targeting the C5 component of complement that silences C5 expression in the liver (ALN-CC5). Treatment of wild-type rodents with ALN-CC5 resulted in robust and durable suppression of liver C5 expression. Dose-dependent serum C5 suppression was observed in non-human primates, with a lowering of serum C5 of up to 97.5% and the concomitant inhibition of serum complement activity. C5 silencing was efficacious in ameliorating disease symptoms in two standard rat models of MG, demonstrating the key role of circulating C5 in pathology at the NMJ. Improvement in disease activity scores and NMJ pathology was observed at intermediate levels of complement activity inhibition, suggesting that complete ablation of complement activity may not be required for efficacy in MG. The pre-clinical studies of ALN-CC5 and efficacy of C5 silencing in rat models of MG support further clinical development of ALN-CC5 as a potential therapeutic for the treatment of MG and other complement-mediated disorders.

Complement components are upregulated and correlate with disease progression in the TDP-43Q331K mouse model of amyotrophic lateral sclerosis

BackgroundComponents of the innate immune complement system have been implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS) specifically using hSOD1 transgenic animals; however, a comprehensive examination of complement expression in other transgenic ALS models has not been performed. This study therefore aimed to determine the expression of several key complement components and regulators in the lumbar spinal cord and tibialis anterior muscle of TDP-43Q331K mice during different disease ages.MethodsNon-transgenic, TDP-43WT and TDP-43Q331K mice were examined at three different ages of disease progression. Expression of complement components and their regulators were examined using real-time quantitative PCR and enzyme-linked immunosorbent assay. Localisation of terminal complement component receptor C5aR1 within the lumbar spinal cord was also investigated using immunohistochemistry.ResultsAltered levels of several major complement factors, including C5a, in the spinal cord and tibialis anterior muscle of TDP-43Q331K mice were observed as disease progressed, suggesting overall increased complement activation in TDP-43Q331K mice. C5aR1 increased during disease progression, with immuno-localisation demonstrating expression on motor neurons and expression on microglia surrounding the regions of motor neuron death. There was a strong negative linear relationship between spinal cord C1qB, C3 and C5aR1 mRNA levels with hind-limb grip strength.ConclusionsThese results indicate that similar to SOD1 transgenic animals, local complement activation and increased expression of C5aR1 may contribute to motor neuron death and neuromuscular junction denervation in the TDP-43Q331K mouse ALS model. This further validates C5aR1 as a potential therapeutic target for ALS.

Specific Inhibition of Complement Activation Significantly Ameliorates Autoimmune Blistering Disease in Mice.

Epidermolysis bullosa acquisita (EBA) is an antibody-mediated blistering skin disease associated with tissue-bound and circulating autoantibodies to type VII collagen (COL7). Transfer of antibodies against COL7 into mice results in a subepidermal blistering phenotype, strictly depending on the complement component C5. Further, activation predominantly by the alternative pathway is required to induce experimental EBA, as blistering was delayed and significantly ameliorated only in factor B−/− mice. However, C5 deficiency not only blocked the activation of terminal complement components and assembly of the membrane attack complex (MAC) but also eliminated the formation of C5a. Therefore, in the present study, we first aimed to elucidate which molecules downstream of C5 are relevant for blister formation in this EBA model and could be subsequently pharmaceutically targeted. For this purpose, we injected mice deficient in C5a receptor 1 (C5aR1) or C6 with antibodies to murine COL7. Importantly, C5ar1−/− mice were significantly protected from experimental EBA, demonstrating that C5a–C5aR1 interactions are critical intermediates linking pathogenic antibodies to tissue damage in this experimental model of EBA. By contrast, C6−/− mice developed widespread blistering disease, suggesting that MAC is dispensable for blister formation in this model. In further experiments, we tested the therapeutic potential of inhibitors of complement components which were identified to play a key role in this experimental model. Complement components C5, factor B (fB), and C5aR1 were specifically targeted using complement inhibitors both prophylactically and in mice that had already developed disease. All complement inhibitors led to a significant improvement of the blistering phenotype when injected shortly before anti-COL7 antibodies. To simulate a therapeutic intervention, anti-fB treatment was first administered in full-blown EBA (day 5) and induced significant amelioration only in the final phase of disease evolution, suggesting that early intervention in disease development may be necessary to achieve higher efficacy. Anti-C5 treatment in incipient EBA (day 2) significantly ameliorated disease during the whole experiment. This finding is therapeutically relevant, since the humanized anti-C5 antibody eculizumab is already successfully used in patients. In conclusion, in this study, we have identified promising candidate molecules for complement-directed therapeutic intervention in EBA and similar autoantibody-mediated diseases.

Liver Transplantation from Female Donors Provokes Higher Complement Component C5a Activity.

BackgroundTransplanted organs from female donors are associated with less favorable prognoses and outcomes. This study aimed to determine whether donor gender affects levels of serum terminal complement component C5a and oxidative stress in pediatric living related liver transplantation (LRLTx) recipients.Material/MethodsThe subjects were 43 patients (20 males and 23 females) who underwent LRLTx during childhood (age range 1.2 years to 14.4 years; mean age 5 years). Serum samples were taken during the patients’ regular outpatient visits after LRLTx. Serum C5a was measured using the specific human C5a ELISA kit. Serum total hydroperoxide (TH) and biological antioxidative potential (BAP) were measured using the free radical analytic system, and the oxidative stress index (OSI) was calculated as the ratio of TH to BAP. Serum glutamic pyruvic transaminase (GPT), glutamic oxaloacetic transaminase (GOT), gamma-glutamyl transpeptidase (γGTP), and lactate dehydrogenase (LDH) were also measured as part of a typical outpatient examination for such patients.ResultsC5a serum levels were higher in the 29 recipients who received their grafted livers from female donors than in the 14 recipients who received their grafted livers from male donors. Recipients who received their grafted livers from female donors had higher incidence of post-LTx (liver transplantation) complications. Female recipients from female donors showed the highest serum GPT and GOT levels, but this difference was only significant when compared to the female recipients from male donors (41.4±9.8 IU/L vs. 17.3±1.8 IU/L for GPT and 42.2±7.5 IU/L vs. 23.4±2.2 IU/L for GOT; P<0.05).ConclusionsPediatric LRLTx patients who receive their grafts from female donors exhibit higher levels of serum C5a that probably plays a role in the immunological response against grafted livers from female donors in LTx.

The role of Lsa23 to mediate the interaction of Leptospira interrogans with the terminal complement components pathway

Leptospirosis is a severe worldwide zoonotic disease caused by pathogenic Leptospira spp. It has been demonstrated that pathogenic leptospires are resistant to the bactericidal activity of normal human serum while saprophytic strains are susceptible. Pathogenic strains have the ability to bind soluble complement regulators and these activities are thought to contribute to bacterial immune evasion. One strategy used by some pathogens to evade the complement cascade, which is not well explored, is to block the terminal pathway. We have, thus, examined whether leptospires are able to interact with components of the terminal complement pathway. ELISA screening using anti-leptospires serum has shown that the pathogenic, virulent strain L. interrogans L1-130 can bind to immobilized human C8 (1 μg). However, virulent and saprophyte L. biflexa strains showed the ability to interact with C8 and C9, when these components were employed at physiological concentration (50 μg/mL), but the virulent strain seemed more competent. Lsa23, a putative leptospiral adhesin only present in pathogenic strains, interacts with C8 and C9 in a dose-dependent mode, suggesting that this protein could mediate the binding of virulent Leptospira with these components. To our knowledge, this is the first work reporting the binding of Leptospira to C8 and C9 terminal complement components, suggesting that the inhibition of this pathway is part of the strategy used by leptospires to evade the innate immunity.

Stopping Eculizumab in Patients with Atypical Hemolytic Uremic Syndrome

Eculizumab, a monoclonal antibody directed against the terminal complement component C5, is approved for the treatment of patients with paroxysmal

Small-molecule factor D inhibitors selectively block the alternative pathway of complement in paroxysmal nocturnal hemoglobinuria and atypical hemolytic uremic syndrome.

Paroxysmal nocturnal hemoglobinuria and atypical hemolytic uremic syndrome are diseases of excess activation of the alternative pathway of complement that are treated with eculizumab, a humanized monoclonal antibody against the terminal complement component C5. Eculizumab must be administered intravenously, and moreover some patients with paroxysmal nocturnal hemoglobinuria on eculizumab have symptomatic extravascular hemolysis, indicating an unmet need for additional therapeutic approaches. We report the activity of two novel small-molecule inhibitors of the alternative pathway component Factor D using in vitro correlates of both paroxysmal nocturnal hemoglobinuria and atypical hemolytic uremic syndrome. Both compounds bind human Factor D with high affinity and effectively inhibit its proteolytic activity against purified Factor B in complex with C3b. When tested using the traditional Ham test with cells from paroxysmal nocturnal hemoglobinuria patients, the Factor D inhibitors significantly reduced complement-mediated hemolysis at concentrations as low as 0.01 μM. Additionally the compound ACH-4471 significantly decreased C3 fragment deposition on paroxysmal nocturnal hemoglobinuria erythrocytes, indicating a reduced potential relative to eculizumab for extravascular hemolysis. Using the recently described modified Ham test with serum from patients with atypical hemolytic uremic syndrome, the compounds reduced the alternative pathway-mediated killing of PIGA-null reagent cells, thus establishing their potential utility for this disease of alternative pathway of complement dysregulation and validating the modified Ham test as a system for pre-clinical drug development for atypical hemolytic uremic syndrome. Finally, ACH-4471 blocked alternative pathway activity when administered orally to cynomolgus monkeys. In conclusion, the small-molecule Factor D inhibitors show potential as oral therapeutics for human diseases driven by the alternative pathway of complement, including paroxysmal nocturnal hemoglobinuria and atypical hemolytic uremic syndrome.

Characterisation and expression analysis of two terminal complement components: C7 and C9 from large yellow croaker, Larimichthys crocea

The large yellow croaker Larimichthys crocea, as one of the most economically important marine fish in China and East Asian countries, are facing the fatal attraction of various pathogens in recent years. Elucidation of the organism immunomodulatory mechanism of croaker response to pathogen infection is essential for the disease control. In present study, we reported for the first time the molecular characterization and expression analysis of two terminal complement components (TCCs) of croaker, Lc-C7 and Lc-C9. These two structural conserved TCCs were detected in many tissues in adult healthy fish, with highest levels detected in liver. The transcriptional expression analysis of Lc-C7 and Lc-C9 at different developmental stages showed a continuous increase towards hatch, however the two TCCs mRNA were not detected at the unfertilized stage, hinting the origination of these two TCCs after fertilization. Rapid and drastic responses to Vibrio alginolyticus challenge were observed for Lc-C7 and Lc-C9, suggesting the involvement of component C7 and C9 in innate immune responses to pathogenic invasion in teleost fish. These findings could deepen our understanding about immunomodulatory mechanisms of croaker and shed a new light to the role of component system in teleostean immunomodulation.

A 425 T>C polymorphism in complement C7 association with resistance to Aeromonas hydrophila in grass carp.

Aeromonas hydrophila, a widespread bacterium in the aquatic environment, causes hemorrhagic septicemia in fish. In the last decade, the disease has caused mass mortalities and tremendous economic loss in cultured fish. The complement component C7 is a terminal component of complement that interacts in a sequence of polymerization reactions with other terminal complement components to form a membrane attack complex. The formation of the membrane attack complex creates a pore in the membranes of certain pathogen that can lead to their death. The objective of this study was to identify single nucleotide polymorphisms (SNPs) in the C7 gene and to assess their association with A. hydrophila resistance in grass carp. A resource population consisting of 186 susceptible and 191 resistant grass carp was constructed. We sequenced a total of 7826 bp of the C7 gene and identified 6 SNPs that were genotyped in the resource population. The SNP -1575 A>C was positioned in the promoter region of the gene. The SNP 425 C>T identified in the coding exon was a synonymous substitution in the fourth exon. Statistical analysis showed that SNP 425 C>T was associated with the incidence of hemorrhagic septicemia. The SNPs -1575 A>C, -688 T>C, and -266 A>C were highly linked together (r(2) > 0.85). No haplotypes generated with these 3 SNPs were associated with resistance to A. hydrophila in grass carp. These findings suggest that the 425 C>T polymorphism in C7 gene may be a significant molecular marker for resistance to A. hydrophila in grass carp.

Complement-mediated 'bystander' damage initiates host NLRP3 inflammasome activation.

Complement activation has long been associated with inflammation, primarily due to the elaboration of the complement anaphylotoxins C5a and C3a. In this work, we demonstrate that the phagocytosis of complement-opsonized particles promotes host inflammatory responses by a new mechanism that depends on the terminal complement components (C5b-C9). We demonstrate that during the phagocytosis of complement-opsonized particles, the membrane attack complex (MAC) of complement can be transferred from the activating particle to the macrophage plasma membrane by a 'bystander' mechanism. This MAC-mediated bystander damage initiates NLRP3 inflammasome activation, resulting in caspase-1 activation and IL-1β and IL-18 secretion. Inflammasome activation is not induced when macrophages phagocytize unopsonized particles or particles opsonized with serum deficient in one of the terminal complement components. The secretion of IL-1β and IL-18 by macrophages depends on NLRP3, ASC (also known as PYCARD) and caspase-1, as macrophages deficient in any one of these components fail to secrete these cytokines following phagocytosis. The phagocytosis of complement-opsonized particles increases leukocyte recruitment and promotes T helper 17 cell (TH17) biasing. These findings reveal a new mechanism by which complement promotes inflammation and regulates innate and adaptive immunity.

Characterization and evolutionary analysis of duplicated C7 in miiuy croaker.

The complement system, as one of the most sophisticated innate immune system, plays an important role in defense against invading microorganisms. The complement component C7 participates in the cytolytic phase of complement activation through a series of polymerization reactions with other terminal complement components. In this study, we derived two C7 genes from the whole genome of miiuy croaker which were the consequence of the fish-specific genome duplication. Our data showed that miiuy croaker C7-1 and C7-2 genes shared same structure domains. The analysis of gene synteny showed that high degree conserved of synteny was retained between miiuy croaker and other teleosts, and miiuy croaker had a relatively closer relationship with fugu. The expression of C7-1 and C7-2 in miiuy croaker healthy tissues revealed that they were ubiquitously expressed in all ten tested tissues. Besides, the immune response of C7-1 and C7-2 were different in spleen with Vibrio anguillarum, Staphylococcus aureus, poly I:C and LPS at 24 h post-injection, respectively. Furthermore, the expression patterns of C7-1 and C7-2 were different in liver, spleen and kidney after infected with V. anguillarum at different time-point. Evolutionary analysis showed that all the ancestral lineages underwent positive selection except for the ancestral lineages of fish C7-2, indicated that the ancestral lineages of fish C7-1 genes undertook more pressures than C7-2 in defense against the invading microorganisms. Meanwhile, a series of maximum likelihood methods were used to explore the evolutionary patterns on extant vertebrates' C7 genes. Three and one positive selection sites were found in extant mammalian C7 genes and fish C7-2 genes, but no positive selection site was found in extant fishes C7-1 genes. The result showed that extant fish C7-2 genes undertook more pressures compared with C7-1. In conclusion, fish C7-1 and C7-2 gene underwent different evolutionary patterns.

Elevation of Circulating Terminal Complement Components in aHUS and TTP: Utility of Genetic Testing to Differentiate Among Thrombotic Microangiopathies

Introduction: The thrombotic microangiopathies (TMA) thrombotic thrombocytopenic purpura (TTP) and atypical hemolytic uremic syndrome (aHUS) are virtually indistinguishable by clinical characteristics. However, their distinct pathophysiology and differential response to plasma exchange (PEx) or anti-C5 complement therapy demands an accurate diagnosis. ADAMTS13 activity <5-10% defines TTP in the majority of instances, but is complicated by the fact that the test is often not ordered prior to institution of PEx, and recent reports of TMAs characterized by ADAMTS13 <5% and high titer IgG that were refractory to PEx but responses to anti-complement therapy complicates the diagnostic dilemma. Elevated levels of terminal complement components (TCCs) C5a and C5b-9 (membrane attack complex, MAC) have been reported in both TTP and aHUS, and recent studies also suggest higher levels of C5b-9, in aHUS compared to TTP. This finding might explain differences in pathophysiology between subtypes of TMAs related to production of TCCs.Objective: We sought to explore the levels of plasma TCCs, including C5a and C5b-9 in TMA subtypes as defined by several different criteria. We hypothesized that they are elevated in both TTP and aHUS-like TMAs, but at significantly higher levels in the latter, and would decline in TMAs in remission, but not normalize.Methods: Blood collected by venipuncture into heparin sodium tubes from acute TMA patients, both primary (no accompanying diagnosis) and secondary (transplant, drug and cancer related cases) (n=44), TMA patients in remission (n=7) and controls. TMA subtypes were categorized by (1) physician clinical diagnoses (TTP n=28, aHUS n=9); (2) ADAMTS13 levels <5-10% (defining TTP, though timing of ADAMTS13 sampling and PEx initiation could not be certified in all cases); (3) initial platelet count and creatinine levels (“TTP” characterized as presenting platelet count <20,000 and/or creatinine <1.7); and (4) presence of germline complement or complement regulatory protein mutations linked to aHUS. TMA remission was defined as platelet count recovery (>150K) and recovery of renal function to baseline off therapy for at least one month. Samples were assayed for C5a and C5b-9 by ELISA. Genomic DNA was isolated from plasmas and amplified by semi-nested PCR using primers to detect complement mutations as we have described previously.Results: We found elevated plasma levels of C5b-9 in acute TMAs compared to adult controls (median 3255 ng/ml; range 772-8109 vs 440; range 218-1136; p= 0.005) and compared to remission TMAs (1638 ng/ml; range 344-2100; p=0.03). When C5b-9 levels were compared between the pre-defined groups, only C5b-9 levels between patients with and without a complement mutations were significant (p=0.014; Figure 1). C5a levels were elevated in all TMAs but could not distinguish between TMA subtypes regardless of definition. Irrespective of diagnosis, levels of C5b-9 decreased from acute TMA levels in remission but still remained elevated compared to cord blood controls (p=0.014).Conclusions: Dysregulation of the alternative complement pathway resulting from genetic deficiency in complement regulatory factors results in elevated TCCs in all TMAs, but absolute levels help to identify a unique subset of aHUS-like TMA patients. Clinical impression, absolute platelet count and creatinine level, or ADAMTS13 activity did not predict the degree of TCC elevation. Elevated C5b-9 in remission TMAs may be related to persistent complement regulation defects. Whether plasma TCC elevation predicts a response to anti-complement therapies remains to be determined. [Display omitted] DisclosuresChapin:Alexion: Honoraria; Novo-Nordisk: Honoraria. Laurence:Alexion : Honoraria, Research Funding, Speakers Bureau; Omeros: Research Funding.

The role of complement in the pathogenesis of renal ischemia-reperfusion injury and fibrosis.

The complement system is a major component of innate immunity and has been commonly identified as a central element in host defense, clearance of immune complexes, and tissue homeostasis. After ischemia-reperfusion injury (IRI), the complement system is activated by endogenous ligands that trigger proteolytic cleavage of complement components via the classical, lectin and/or alternative pathway. The result is the formation of terminal complement components C3a, C5a, and the membrane attack complex (C5b-9 or MAC), all of which play pivotal roles in the amplification of the inflammatory response, chemotaxis, neutrophil/monocyte recruitment and activation, and direct tubular cell injury. However, recent evidence suggests that complement activity transcends innate host defense and there is increasing data suggesting complement as a regulator in processes such as allo-immunity, stem cell differentiation, tissue repair, and progression to fibrosis. In this review, we discuss recent advances addressing the role of complement as a regulator of IRI and renal fibrosis after organ donation for transplantation. We will also briefly discuss currently approved therapies that target complement activity in kidney ischemia-reperfusion and transplantation.