Terminal Components Research Articles

Isolation and characterization of a recombinant class C acid phosphatase from Sphingobium sp. RSMS strain

Tributyl phosphate (TBP) is extensively used in nuclear industry and is a major environmental pollutant. The mechanism for TBP degradation is not identified in any TBP-degrading bacteria. Here, we report identification of an acid phosphatase from Sphingobium sp. RSMS (Aps) that exhibits high specific activity towards monobutyl phosphate (MBP) and could be a terminal component of the TBP degradation process. A genomic DNA library of the bacteria was screened using a histochemical method which yielded 35 phosphatase clones. Among these, the clone that showed the highest MBP degradation was studied further. DNA sequence analysis showed that the genomic insert encodes a protein (Aps) which belongs to class C acid phosphatase. The recombinant Aps was found to be a dimer and hydrolysed MBP with a Kcat 68.1 ± 5.46 s−1 and Km 2.5 mM ± 0.50. The protein was found to be nonspecific for phosphatase activity and hydrolyzed disparate organophosphates.

Genetic workup as a complementary tool for the diagnosis of primary complement component deficiencies: a multicenter experience.

Awareness of risk factors for primary complement deficiencies, even at the first infectious episode, should facilitate prompt immune and genetic workup, commencing diagnosis and proper treatment for the patient and family. •Deficiencies in the classical terminal complement components increase susceptibility to invasive meningococcal infections. • Recurrent meningococcal infections mandate a diagnostic workup of the complement system. •Genetic workup can be utilized for prompt diagnosis of complement deficiencies. •High rates of consanguinity, even in the presence of a single meningococcal infection, should promote immune and genetic workups.

Echo reception in group flight by Japanese horseshoe bats, Rhinolophus ferrumequinum nippon.

The ability to detect behaviourally relevant sensory information is crucial for survival. Especially when active-sensing animals behave in proximity, mutual interferences may occur. The aim of this study was to examine how active-sensing animals deal with mutual interferences. Echolocation pulses and returning echoes were compared in spaces of various sizes (wide and narrow) in Rhinolophus ferrumequinum nippon flying alone or in a group of three bats. We found that in the narrow space, the group-flying bats increased the duration and bandwidth of the terminal frequency-modulated component of their vocalizations. By contrast, the frequency of the returning echoes did not differ in the presence of conspecifics. We found that their own echo frequencies were compensated within the narrow frequency ranges by Doppler shift compensation. By contrast, the estimated frequencies of the received pulses emitted by the other bats were much more broadly distributed than their echoes. Our results suggest that the bat auditory systems are sharply tuned to a narrow frequency to filter spectral interference from other bats.

The importance of terminal complement inhibition in paroxysmal nocturnal hemoglobinuria.

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare, chronic hematologic disorder associated with inappropriate terminal complement activity on blood cells that can result in intravascular hemolysis (IVH), thromboembolic events (TEs), and organ damage. Untreated individuals with PNH have an increased risk of morbidity and mortality. Patients with PNH experiencing IVH often present with an elevated lactate dehydrogenase (LDH; ⩾ 1.5 × the upper limit of normal) level which is associated with a significantly higher risk of TEs, one of the leading causes of death in PNH. LDH is therefore used as a biomarker for IVH in PNH. The main objective of PNH treatment should therefore be prevention of morbidity and mortality due to terminal complement activation, with the aim of improving patient outcomes. Approval of the first terminal complement inhibitor, eculizumab, greatly changed the treatment landscape of PNH by giving patients an effective therapy and demonstrated the critical role of terminal complement and the possibility of modulating it therapeutically. The current mainstays of treatment for PNH are the terminal complement component 5 (C5) inhibitors, eculizumab and ravulizumab, which have shown efficacy in controlling terminal complement-mediated IVH, reducing TEs and organ damage, and improving health-related quality of life in patients with PNH since their approval by the United States Food and Drug Administration in 2007 and 2018, respectively. Moreover, the use of eculizumab has been shown to reduce mortality due to PNH. More recently, interest has arisen in developing additional complement inhibitors with different modes of administration and therapeutics targeting other components of the complement cascade. This review focuses on the pathophysiology of clinical complications in PNH and explores why sustained inhibition of terminal complement activity through the use of complement inhibitors is essential for the management of patients with this chronic and debilitating disease.

A modified pseudo 2D physically-based model for double-gate TFETs: Role of precise calculations of drain and source depletion regions

In this current study, a modified pseudo two-dimensional (2-D) semi-analytical model for double gate tunnel FETs (DG-TFETs) is introduced. The main regions in the DG-TFET structure are the channel and the depletion regions inside the source and the drain. In such regions, the 2-D Poisson's equation is solved by adjusting suitable boundary conditions. The source and the drain depletion region lengths are calculated precisely by an iterative technique resulting in an accurate prediction of the electrostatic potential. According to the obtained 2-D electrostatic potential, the energy band diagram could be extracted and, consequently, the minimum tunneling width is calculated. Accordingly, the model of drain current is introduced by applying Kane's tunneling model. Further, the source depletion charge and the channel charge are derived, and the terminal capacitance components are then extracted. A comparison between the proposed model and SILVACO TCAD simulations shows a satisfactory agreement that confirms the validation of the presented model robustness.

Abstract PO-196: Upregulation of bacterial and fungal pathogen sensing genes in preinvasive colorectal lesions in African Americans compared to Caucasian Americans

Abstract Background: Colorectal cancer (CRC) incidence and mortality are higher for African Americans (AAs) than Caucasian Americans (CAs). Variation in tumor immune responses by race appears to be important for prognosis. However, few studies have compared immune gene expression in preinvasive lesions where prevention may be possible. Methods: The immune gene expression of 95 cases of colorectal adenomas (AA 47, CA 48) excised via sigmoidoscopy or colonoscopy with polypectomy between October 2012 and May 2016 was assessed using the NanoString nCounter platform using the immunology v2 panel (n=579 genes). Data were analyzed using a NanoStringDiff R package, implementing negative binomial regression models of gene expression as the dependent variable and race group (AA vs. CA) as the primary predictor variable. Models were adjusted for age, sex, location within the colon, histology, and degree of dysplasia. Results: In AAs vs. CAs, 24 immune genes were up-regulated, while 11 genes were down-regulated (for each gene, p<0.01). Compared to CAs, genes overexpressed in AAs suggest more robust antibacterial and antifungal responses (e.g., IL17F, IL21, AIRE, IL1A, GATA3, MARCO, FCGR1A). Enhanced expression of terminal components of the complement pathway C8B and C9 further support an increased effector response in AAs. Additionally, genes related to wound healing (e.g., GP1BB, XCL1) were overexpressed in AAs. Genes associated with stimulation of cytotoxic responses (NCR1, HLADRB1), complement activation through lectin-related pathways (ITLN2, MBL2), and recruitment and function of innate immune cells (IL8 and IL6) had lower expression in AAs vs. CAs. Conclusion: Our results suggest that preinvasive neoplasms in AAs vs. CAs display a gene expression that is suggestive of heightened sensing of bacterial and fungal commensals, which may potentially inflame local tissues and support tumor progression. Understanding the molecular differences in early tumorigenesis between AAs vs. CAs may help inform prevention interventions to address racial disparities. Citation Format: Silvia Guglietta, Lauren R. Fanning, Alexandria G. Cousart, Kent E. Armeson, Zequn Sun, David N. Lewin, J Grant Brazeal, Dongjun Chung, Alexander V. Alekseyenko, Kristin Wallace. Upregulation of bacterial and fungal pathogen sensing genes in preinvasive colorectal lesions in African Americans compared to Caucasian Americans [abstract]. In: Proceedings of the AACR Virtual Conference: 14th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2021 Oct 6-8. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr PO-196.

Динамическое программирование в задаче маршрутизации: декомпозиционный вариант

The questions of applying the dynamic programming (DP) apparatus to the routing problem with constraints and cost functions with the tasks list dependence are investigated. It is supposed that binary partition of the task set is given; tasks of the first task group must be fulfilled before the fulfillment of the task of the second group begins. In each of the groups, precedence conditions may be present. This setting can be applied in the case of sheet cutting on CNC machines, where two above-mentioned groups form zones planned at the cutting stage. In general case, for the optimal solution construction, the two-stage variant of DP is used. Linking two versions of DP is realized by identification of the criterion terminal component for service problem of the first group with extremum function connected with the second group. The connection of optimal solutions for above-mentioned two problems allows to construct an optimal solution for the initial joint problem. Based on the theoretical constructions algorithm realized on personal computer is constructed; computing experiment is realized.

Microstructure and Lamellae Phase of Raw Natural Rubber via Spontaneous Coagulation Assisted by Sugars.

Natural rubber (NR) as a renewable biopolymer is often produced by acid coagulation of fresh natural latex collected from Hevea brasiliensis. However, this traditional process is facing a huge economic and environmental challenge. Compared with the acid coagulation, spontaneous or microorganism coagulation is an ecofriendly way to obtain NR with excellent performance. To clarify the influence of different sugars on NR quality, several sugars were used to assist the coagulation process. Influence of different sugars on microstructure and cold crystallization were examined by 1H NMR, DSC, etc. The results indicated that sugars exhibit different biological activity on terminal components of fresh field latex and can influence the resultant molecular structure and basic properties. Brown sugar exhibits higher metabolic activity and is inclined to decompose the protein and phospholipids crosslinking compared with other sugars. The larger molecular weight of sugar molecule is beneficial for the formation of the stable α lamellae phase and higher overall degree of crystallization.

P-Wave Morphology in Focal Atrial Tachycardia: An Updated Algorithm to Predict Site of Origin.

This study sought to report P-wave morphology (PWM) from a series of paraseptal (PS) atrial tachycardia (AT), revise then prospectively evaluate a simplified PWM algorithm against a contemporary consecutive cohort with focal AT. The 2006 PWM algorithm was useful in predicting the origin of focal AT. An updated algorithm was developed given advances in multipolar 3-dimensional mapping, potential limitations of PWM in separating PS sites, and a renewed interest in the P-wave in mapping non-pulmonary vein triggers. The PWM from a consecutive series of 67 patients with PS AT were analyzed. PS sites included were coronary sinus ostium, perinodal, left and right septum, septal tricuspid annulus, superior mitral annulus, and noncoronary cusp. Next the P-wave algorithm was revised and prospectively evaluated by 3 blinded assessors. The P-wave for PS sites was neg/pos (n= 50), iso/pos (n= 10), or isoelectric (n= 4) in lead V1 (96%). The P-wave algorithm was modified and prospectively applied to 30 consecutive patients with focal AT who underwent successful ablation. Foci (n=30) originated from the right atrium (33.3%), left atrium (30%), and PS (36.7%). Using the PW algorithm, the correct anatomic location was identified in 93%. Incorrect interpretation of the terminal positive P-wave component (n=3) and initial negative P-wave deflection (n=1) in lead V1 misidentified 4 paraseptal cases. The revised PWM algorithm offers a simplified and accurate method of localizing the responsible site for focal AT. The P-wave remains an important first step in mapping atrial arrhythmias.

Cytokine and Complement Response in the Glaucomatous βB1-CTGF Mouse Model.

Glaucoma is a complex neurodegenerative disease leading to a loss of retinal ganglion cells (RGCs) and optic nerve axons. An activation of the complement system seems to contribute to cell loss in this disease. Hence, we investigated a possible initiation of the complement system and the cytokine response in the βB1-CTGF glaucoma model. In these mice, intraocular pressure is elevated, which is the main glaucoma risk factor in patients, and RGC loss occurs at 15 weeks of age. Therefore, quantitative real-time PCR and immunohistological experiments were performed in 5-, 10-, and 15-week-old βB1-CTGF animals and their corresponding wildtypes (WT) to analyze the expression of several complement system factors. We could show that mRNA levels of the terminal complement pathway components C3 and C5 (Hc) were upregulated at 10 weeks. In accordance, more C3+ and membrane attack complex+ cells were observed in transgenic retinae. Further, the C5a receptor anaphylatoxin receptor (C5ar) and the complement component C5a receptor 1 (C5ar1; CD88) mRNA levels were upregulated in 10- and 15-week-old βB1-CTGF mice. Interestingly, all three activation routes of the complement system were elevated in βB1-CTGF mice at some age. Especially C1q, as a marker of the classical pathway, was significantly increased at all investigated ages. Furthermore, mRNA expression levels of interferon-γ (Infg) were upregulated at 5 weeks, while Cxcl1 and Cxcl2 mRNA levels were upregulated at 10 and 15 weeks. The mRNA levels of the chemokines Cxcl10 were increased at all ages in βB1-CTGF mice. These results lead to the assumption that in these transgenic mice, a complement activation mainly through the classical pathway as well as a cytokine response plays a major role in cell death.

A Challenging Case of Atypical Hemolytic Uremic Syndrome in a Young African American Patient

BackgroundIt is known that malignant hypertension (mHTN) and thrombotic microangiopathy (TMA) commonly coexist. Deciding which phenomenon preceded the other remains a clinical dilemma, specifically in African American patients. However, making that determination is of utmost importance because the management will be different, and that can have dramatic effects on prognosis and outcomes. Herein, we report a case of atypical hemolytic uremic syndrome (aHUS) presenting as mHTN.Case PresentationA 35-year-old African American male with known history of hypertension, presented with nausea, vomiting, and diarrhea for four days. He also reported fatigue and exertional shortness of breath. Upon presentation, his blood pressure was 260/160 mmHg, otherwise physical exam was unremarkable. Initial work up showed hemoglobin of 8.8 g/dL (baseline 13.5), platelet count of 21,000/mL (baseline 250,000), serum creatinine of 16.99 mg/dL (baseline 0.99), MCV (84 fl), increased reticulocyte production index (3.58), increased LDH (1709 U/L), undetectable haptoglobin, and numerous schistocytes on peripheral blood smear. He was admitted as a case of hypertensive emergency and TMA. IV labetalol and hemodialysis were started. Given his gastrointestinal symptoms; stool for Shigella and E.Coli O157:H7 were checked and they were negative.Given the severity of his hematologic derangements and difficult to control blood pressure, we decided to proceed with renal biopsy to rule out primary aHUS which showed thrombotic microangiopathy, global glomerulosclerosis, moderate interstitial fibrosis and tubular atrophy suggestive of aHUS or rheumatologic disorders like systemic sclerosis and arguing against malignant HTN as the sole player. ANA and anti-Scl-70 antibodies were negative. Final impression was aHUS by exclusion, and patient received meningococcal vaccines (Menactra and Bexsero) in preparation to start eculizumab. aHUS genetic panel was sent which came back equivocal as it showed mutations of unknown significance (homozygous missense mutation in the MASP2 gene and 2 heterozygous mutations in the C2 gene).He was started on eculizumab 900 mg weekly for 4 weeks then 1200 mg biweekly starting week 5. He was seen in the office 2 months after initial presentation and receiving 5 doses of Eculizumab. His kidney function showed improvement with > 2 liters of urine output daily, blood pressure was better controlled. A decision by nephrology was made to give him a break from dialysis and remains dialysis-free a year later.DiscussionaHUS is a rare disorder with an estimated prevalence of seven per one million children in Europe. It causes uninhibited activation of complement factors that leads to renal endothelial damage and activation of coagulation cascade leading to TMA. The diagnosis of aHUS requires the fulfillment of the classical triad (microangiopathic hemolytic anemia, thrombocytopenia, and acute renal failure) with a positive gene mutation or antibodies to complement factors. However, absence of these mutations or antibodies, as in the presented case, do not exclude the diagnosis. The early diagnosis of aHUS is necessary for treatment with eculizumab, a monoclonal antibody against C5 to block the terminal complement cascade. Kidney biopsy can be helpful in equivocal cases especially if it shows only the typical changes of malignant hypertension which essentially rules out aHUS. Hypertension with concurrent TMA is treated with strict BP control which is often enough to resolve TMA features and restores renal function, at least partially. On the contrary, aHUS causing severe HTN needs more sophisticated testing and blockade of the terminal complement component to improve outcome; that's why the distinction of which one is the primary process is of utmost importance. Our case emphasizes the importance of having low threshold for testing for aHUS in patients with mHTN and TMA, especially in African American patients where malignant HTN is known to happen more commonly, and to notice the subtle hints that may help in this distinction, such as profound hemolysis or thrombocytopenia out of proportion to what one would expect from mHTN alone. Early recognition of aHUS may save a patient's kidney. DisclosuresNo relevant conflicts of interest to declare.

Eculizumab Pharmacokinetics and Pharmacodynamics in Patients With Neuromyelitis Optica Spectrum Disorder.

Objective: To investigate the pharmacokinetics and pharmacodynamics of the approved 900/1,200 mg dosing regimen for the terminal complement component 5 (C5) inhibitor eculizumab in patients with neuromyelitis optica spectrum disorder (NMOSD).Methods: Data were analyzed from 95 patients with aquaporin-4-IgG-positive NMOSD who received eculizumab during the PREVENT study (ClinicalTrials.gov: NCT01892345). Relationships were explored between eculizumab exposure and free complement C5 concentrations, terminal complement activity, and clinical outcomes.Results: Pharmacokinetic data were well-described by a two-compartment model with first-order elimination, and time-variant body-weight and plasmapheresis/plasma exchange effects. Steady-state serum eculizumab concentrations were achieved by Week 4 and were sustained, with serum trough eculizumab concentrations maintained above the 116 μg/ml threshold for complete complement inhibition throughout 168 weeks of treatment in all post-baseline samples from 89% of patients. Complete inhibition of terminal complement was achieved at Day 1 peak and pre-dosing trough eculizumab concentration in nearly all post-baseline samples assessed (free C5 <0.5 μg/ml in all post-baseline samples from 96% of patients; in vitro hemolysis <20% in all post-baseline samples from 93% of patients). Kaplan–Meier survival analysis of time to first relapse showed separation of eculizumab-treated patients from those receiving placebo, but no separation based on eculizumab exposure quartile, indicating an optimized dose regimen with maximized efficacy.Conclusions: The approved eculizumab dosing regimen (900/1,200 mg) for adults with aquaporin-4-IgG-positive NMOSD is confirmed by rigorous quantitative model-based analysis of exposure–response. The data demonstrate that eculizumab's mechanism of action translates into clinical effect by achieving rapid, complete, and sustained terminal complement inhibition.

Eculizumab Pharmacokinetics and Pharmacodynamics in Patients With Generalized Myasthenia Gravis.

Objective: To investigate the pharmacokinetics, pharmacodynamics, and exposure–response of the approved 900/1,200 mg dosing regimen for the terminal complement component 5 (C5) inhibitor eculizumab in patients with generalized myasthenia gravis (gMG).Methods: The analysis used data from 62 patients aged ≥ 18 years with anti-acetylcholine receptor (AChR) antibody-positive refractory gMG who received eculizumab during the REGAIN study (ClinicalTrials.gov: NCT01997229). One- and two-compartment population-pharmacokinetic models were evaluated, and the impact of covariates on pharmacokinetic parameters was assessed. Relationships between eculizumab exposure and free C5 concentration, in vitro hemolytic activity, clinical response, and tolerability were characterized.Results: Steady-state serum eculizumab concentrations were achieved by Week 4 and were sustained throughout the 26-week treatment period. The eculizumab pharmacokinetic data were well-described by a two-compartment model with first-order elimination, including effects of body weight on pharmacokinetic parameters and plasma-exchange events on clearance. Complete inhibition of terminal complement was achieved in nearly all patients at the time of trough and peak eculizumab concentrations at all post-dose timepoints assessed (free C5 < 0.5 μg/ml in 92% of patients; in vitro hemolysis < 20% in 87% of patients). Serum eculizumab concentrations of ≥116 μg/ml achieved free C5 concentrations of < 0.5 μg/ml. Clinical efficacy and tolerability were consistent across the eculizumab exposure range.Conclusions: Rigorous, quantitative, model-based exposure–response analysis of serum eculizumab concentration and response data demonstrated that the approved eculizumab dosing (900/1,200 mg) for adults with anti-AChR antibody-positive refractory gMG rapidly achieved complete inhibition of terminal complement activation and provided sustained clinical efficacy across the eculizumab exposure range.

Computational image enhancement of multimode fibre-based holographic endo-microscopy: harnessing the muddy modes.

In imaging geometries, which employ wavefront-shaping to control the light transport through a multi-mode optical fibre (MMF), this terminal hair-thin optical component acts as a minimally invasive objective lens, enabling high resolution laser-scanning fluorescence microscopy inside living tissues at depths hardly accessible by any other light-based technique. Even in the most advanced systems, the diffraction-limited foci scanning the object across the focal plane are contaminated by a stray optical signal carrying typically few tens of % of the total optical power. The stray illumination takes the shape of a randomised but reproducible speckle, and is unique for each position of the focus. We experimentally demonstrate that the performance of imaging a fluorescent object can be significantly improved, when resulting images are computationally post-processed, utilising records of intensities of all speckle-contaminated foci used in the imaging procedure. We present two algorithms based on a regularised iterative inversion and regularised direct pseudo-inversion respectively which lead to enhancement of the image contrast and resolution.

Complement C3 mediates early hippocampal neurodegeneration and memory impairment in experimental multiple sclerosis

Memory impairment is one of the disabling manifestations of multiple sclerosis (MS) possibly present from the early stages of the disease and for which there is no specific treatment. Hippocampal synaptic dysfunction and dendritic loss, associated with microglial activation, can underlie memory deficits, yet the molecular mechanisms driving such hippocampal neurodegeneration need to be elucidated. In early-stage experimental autoimmune encephalomyelitis (EAE) female mice, we assessed the expression level of molecules involved in microglia-neuron interactions within the dentate gyrus and found overexpression of genes of the complement pathway. Compared to sham immunized mice, the central element of the complement cascade, C3, showed the strongest and 10-fold upregulation, while there was no increase of downstream factors such as the terminal component C5. The combination of in situ hybridization with immunofluorescence showed that C3 transcripts were essentially produced by activated microglia. Pharmacological inhibition of C3 activity, by daily administration of rosmarinic acid, was sufficient to prevent early dendritic loss, microglia-mediated phagocytosis of synapses in the dentate gyrus, and memory impairment in EAE mice, while morphological markers of microglial activation were still observed. In line, when EAE was induced in C3 deficient mice (C3KO), dendrites and spines of the dentate gyrus as well as memory abilities were preserved. Altogether, these data highlight the central role of microglial C3 in early hippocampal neurodegeneration and memory impairment in EAE and, therefore, pave the way toward new neuroprotective strategies in MS to prevent cognitive deficit using complement inhibitors.

Historical aspects of muscle research in the Dubowitz Neuromuscular Centre: the Hammersmith days

Historical aspects of muscle research in the Dubowitz Neuromuscular Centre: the Hammersmith days

On sequential traversal of sets

The problem of sequential traversal of megapolises with precedence conditions is investigated; this problem is oriented to mechanical engineering — CNC metal cutting machines. There is the following setting singularity: the terminal component of additive criterion contains the dependence on the starting point. This singularity leads to the fact that the natural solution procedure based on dynamic programming must be applied individually for every starting point. The investigation goal consists in the construction of an optimizing algorithm for determining a complex including a route (a variant of megapolis numbering), a trajectory, and a starting point. The proposed algorithm realizes an idea of directed enumeration of starting points. This algorithm is realized as a program for PC; computations for model examples are made.

PH-sensitive and tumor-targeting nanogels based on ortho ester-modified PEG for improving the in vivo anti-tumor efficiency of doxorubicin

In this study, we aim to develop the pH-sensitive and tumor-targeting nanogels based on the co-polymerization of three terminal allyl-functionalized components, including ortho ester-conjugated mPEG (mPEG-MOE), ortho ester crosslinker (OEAM) and phenylboronic acid (APBA). The hybrid nanogels displayed a typical spherical structure with a diameter around 200 nm observed by dynamic light scattering (DLS) and scanning electron microscopy (SEM). The prepared nanogels possessed a good stability in neutral conditions, while displayed pH-triggered drug release profiles. Furthermore, in vitro study of cellular uptake and cytotoxicity indicated that the nanogels possessed the highest drug accumulation and cytotoxicity against EMT6 cells. In vivo antitumor examination suggested that these nanogels brought out excellent efficacy in enhancing drug concentration, restraining tumor growth, and prolonged the survival time of tumor-bearing mice. Thus, the prepared multi-functional nanogels possess great potentials for drug delivery in tumor treatment.

Similarities and differences in the structures and proteoform profiles of the complement proteins C6 and C7

The human complement system provides a first line of defence against pathogens. It requires a well-orchestrated sequential assembly of an array of terminal complement components (C5, C6, C7, C8, and C9), ultimately forming the membrane attack complex (MAC). Although much information about MAC assembly is available, the structure of the soluble C7 has remained elusive. The complement proteins C7 and C6 share very high sequence homology and exhibit several conserved domains, disulphide bridges, and C-mannosylation sites. Here, we used an integrative structural MS-based approach combining native MS, glycopeptide-centric MS, in-gel cross-linking MS (IGX-MS) and structural modelling to describe structural features, including glycosylation, of human serum soluble C7. We compare this data with structural and glycosylation data for human serum C6. The new structural model for C7 shows that it adopts a compact conformation in solution. Although C6 and C7 share many similarities, our data reveals distinct O-, and N-linked glycosylation patterns in terms of location and glycan composition. Cumulatively, our data provide valuable new insight into the structure and proteoforms of C7, solving an essential piece of the puzzle in our understanding of MAC assembly.

In Vitro Combination Studies of ACH-4471 with Eculizumab to Assess a Potential “Switch” Treatment Approach for Paroxysmal Nocturnal Hemoglobinuria

Introduction: ACH-4471 is an oral complement factor D (fD) inhibitor that has demonstrated clinical efficacy in an ongoing phase 2 clinical study for naïve patients suffering from paroxysmal nocturnal hemoglobinuria (PNH). PNH is a rare clonal blood disorder in which erythrocytes lacking the GPI-anchored complement regulators CD55 and CD59 are susceptible to destruction by the terminal complement complex (also known as membrane attack complex, MAC) following the normal low-level activation of the complement alternative pathway (AP) in the fluid phase. The therapeutic agent eculizumab, a monoclonal antibody directed against the terminal complement component C5, blocks MAC assembly on erythrocyte membranes and so prevents most intravascular hemolytic destruction. Eculizumab treatment however leads to increased deposition of complement C3 fragments on PNH membranes, which can result in extravascular phagocytic elimination of opsonized erythrocytes (A. M. Risitano et al, Blood, 2009, 113:4094) and incomplete inhibition of intravascular hemolysis (M. J. Harder et al, Blood, 2017, 129:970), and consequently to the continued anemia and transfusion dependence observed in a significant subset of patients. As an AP inhibitor, in contrast to eculizumab, ACH-4471 prevents both MAC assembly and C3 fragment deposition on PNH cell surfaces and therefore is expected to address the medical needs of these suboptimal responders to eculizumab. To assess this notion directly and also to support a future switch treatment approach, a further clinical study has been planned to evaluate ACH-4471 co-administration in PNH patients who respond suboptimally to eculizumab. In the present study, we conducted in vitro combination experiments to explore the potential pharmacological consequences when ACH-4471 is added on to eculizumab or another anti-C5 monoclonal antibody, using functional AP assays that include the hemolysis of erythrocytes from a PNH patient.Methods: Concentration series of ACH-4471 were assessed in pairwise combinations with eculizumab and a second monoclonal antibody inhibitor of complement C5. Functional assays for human serum AP activity included a single-plate ELISA-based functional assay (Wieslab) and the hemolysis of red blood cells from a PNH patient under AP-restricted conditions but at neutral pH. Analyses of interactions were performed using a three-dimensional surface graphing method and, where applicable, a median-effect plot method.Results: We assessed pairwise interactions using two assays for AP function. In the AP Wieslab assay, interactions between ACH-4471 and an anti-C5 monoclonal antibody ranged from additive to moderately synergistic. Next, to better approximate the physiological setting, we assessed inhibitor interactions against the AP-mediated hemolysis of red blood cells obtained from a PNH patient. ACH-4471 in this assay showed strongly synergistic interactions when tested with eculizumab. In light of a recent report on incomplete inhibition by eculizumab following strong complement activation, we propose a possible mechanism for synergy between ACH-4471 and eculizumab wherein ACH-4471 reduces the density of deposited C3b clusters, to which C5 binding is thereby more readily inhibited by eculizumab.Conclusions: ACH-4471 showed strong synergy with eculizumab. These results suggest the potential clinical utility of ACH-4471 as a PNH therapeutic agent that may be used in suboptimal eculizumab responders in support of a “switch” treatment strategy. DisclosuresPatel:Achillion: Employment. Thanassi:Achillion: Employment. Yang:Achillion: Employment. Brodsky:Alexion Pharmaceuticals, Inc.: Membership on an entity's Board of Directors or advisory committees, Other: Grant Funding. Huang:Achillion: Employment. Podos:Achillion: Employment.