Human C3 Research Articles

Pozelimab, a Human Monoclonal Antibody Against Complement Factor C5, Provided Inhibition of Intravascular Hemolysis in Patients with Paroxysmal Nocturnal Hemoglobinuria

INTRODUCTIONParoxysmal Nocturnal hemoglobuinuria (PNH) is a rare, acquired, life-threatening disorder characterised by intravascular hemolysis and increased risk of thrombosis. Current available treatments represent a significant burden to patients and include anti-complement factor 5 (C5) therapies, eculizumab and ravulizumab, both intravenous infusions, or more recently, twice weekly C3 inhibitor, Pegcetacoplan, a subcutaneous (SC) infusion treatment.Pozelimab (REGN3918), a fully human monoclonal immunoglobulin G4 antibody directed against C5, has been shown to bind with high affinity to wild-type and variant (R885H/C) human C5 and block its activity.In a healthy volunteer study (NCT03115996), SC administration of pozelimab was found to be generally well tolerated while providing complete inhibition of ex vivo-assessed hemolytic activity [Weyne J et al. Blood. 2018;S1:1039]. The data suggested that pozelimab may provide control of intravascular hemolysis in patients with active PNH, and thus could provide an important new alternative for patients supporting progression to a first-in-patient study of pozelimab in patients with active PNH.OBJECTIVETo demonstrate a clinically significant reduction in intravascular hemolysis by once-weekly SC administration of pozelimab over 26 weeks of treatment in patients with active PNH.METHODSThis is a phase 2, open-label, single-arm, 26-week treatment study in 24 patients with active symptomatic PNH who are naïve to complement inhibitor therapy, or who have received prior treatment with a complement inhibitor but not within 6 months prior to the start of the study (NCT03946748).The treatment regimen consists of pozelimab as a single IV loading dose of 30 mg/kg followed thereafter by weekly SC 800 mg administrations.The effect of pozelimab on intravascular hemolysis (monitored via LDH levels) and transfusion avoidance, as well as safety, is to be assessed from baseline to week 26 (study day 183; only partial data were available for some patients at the time of abstract submission). Pozelimab pharmacodynamics was assessed utilizing a sheep red blood cell (RBC) complement activity assay (CH50) and rabbit RBC complement activity assay (AH50).RESULTSAll 24 patients completed the 26-week treatment period with no study drug discontinuation and have been enrolled into an ongoing open-label extension study (NCT04162470). An interim analysis on the first 17 patients was previously reported. All 17 patients had at least 71 days of treatment, with 10 patients receiving treatment for up to 183 days. All enrolled patients had baseline LDH levels ≥2 x upper limit of normal (ULN). Participants were enrolled in two cohorts: Cohort A for dose confirmation and Cohort B for further evaluation of efficacy and safety.Interim Efficacy AnalysisAs previously reported, treatment with pozelimab led to a rapid and sustained reduction in LDH through study week 26. All 17 patients achieved LDH reduction to below the clinically significant threshold of LDH ≤1.5 x ULN.All but one patient achieved control of intravascular hemolysis (LDH ≤1.5 x ULN) at week 2, and all but one patient achieved normalization of LDH (LDH ≤1.0 x ULN) at week 4.Importantly, one patient who is a carrier of a C5 variant known to be resistant to blockade by eculizumab/ravulizumab demonstrated rapid and sustained normalization of LDH.Interim SafetyAs previously reported, no serious adverse events or adverse events leading to treatment discontinuation were reported. Common treatment-emergent adverse events included headache (4 patients; 23.5%) and nausea (2 patients; 11.8%).No breakthrough hemolysis events were observed.CONCLUSIONSPozelimab administered SC once weekly led to the inhibition of intravascular hemolysis in patients with active PNH and was generally well-tolerated. An update to the previously reported interim analysis will be provided.Upon successful completion of the 26-week treatment period, patients are enrolled into an open-label extension study. DisclosuresWeyne: Regeneron Pharmaceuticals, Inc.: Current Employment, Current holder of stock options in a privately-held company. Chaudhari: Regeneron Pharmaceuticals, Inc.: Current Employment, Current holder of stock options in a privately-held company. Harari: Regeneron Pharmaceuticals, Inc.: Current Employment, Current holder of stock options in a privately-held company. Miller: Regeneron Pharmaceuticals, Inc.: Current Employment, Current holder of stock options in a privately-held company. Dain: Regeneron Pharmaceuticals, Inc.: Other: Contractor. Meagher: Regeneron Pharmaceuticals, Inc.: Current Employment, Current holder of stock options in a privately-held company. Rodgers: Regeneron Pharmaceuticals, Inc.: Current Employment, Current holder of stock options in a privately-held company. Perlee: Regeneron Pharmaceuticals, Inc.: Current Employment, Current holder of stock options in a privately-held company. Morton: Regeneron Pharmaceuticals, Inc.: Current Employment, Current holder of stock options in a privately-held company. Weinreich: Regeneron Pharmaceuticals, Inc.: Current Employment, Current holder of stock options in a privately-held company. Yancopoulos: Regeneron Pharmaceuticals, Inc.: Current Employment, Current holder of stock options in a privately-held company. Griffin: Alexion, AstraZeneca Rare Disease: Honoraria, Membership on an entity's Board of Directors or advisory committees; BioCryst Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Sobi Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees; Apellis: Other: Educational grant support.

A Randomized, Double-Blind, Single-Dose Phase 1 Comparative Pharmacokinetic Study Comparing SB12 (Eculizumab Biosimilar) with Reference Eculizumab in Healthy Volunteers

Background: SB12 has been developed as a biosimilar of the reference product (RP) eculizumab. Eculizumab is a humanized monoclonal antibody (IgG2/4 kappa immunoglobulin) that binds to the human C5 complement protein with high affinity. Binding to this protein blocks its cleavage into C5a and C5b, thereby inhibiting terminal complement-mediated intravascular haemolysis. It is currently indicated for the treatment of patients with paroxysmal nocturnal haemoglobinuria, atypical haemolytic uremic syndrome (aHUS), refractory generalized Myasthenia Gravis (gMG) and Neuromyelitis Optica Spectrum Disorder (NMOSD).Objectives: To demonstrate pharmacokinetic (PK) equivalence and evaluate pharmacodynamic (PD), safety, tolerability, and immunogenicity profiles between SB12 and the RP eculizumab.Methods: This was a double-blind, three-arm, parallel group, and single-dose study in healthy subjects, between 18-55 years of age, randomized in a ratio of 1:1:1 to receive a single 300 mg dose of either SB12, European Union (EU) sourced eculizumab, or United States of America (US) sourced eculizumab via intravenous (IV) infusion for 35 minutes. Blood samples for PK and PD analysis were collected over 64 days. The primary objective of this study was to demonstrate PK similarity between the investigational products (IPs), as assessed by area under the concentration-time curve from time zero to infinity (AUC inf). Secondary PK endpoints included area under the concentration-time curve from time zero to the last quantifiable concentration (AUC last) and maximum observed concentration (C max). Equivalence for the primary endpoint (AUC inf) was determined if 90% confidence intervals (CIs) for the ratio of geometric least squared means (LSMeans) of SB12 to EU sourced eculizumab, SB12 to US sourced eculizumab, and EU sourced eculizumab to US sourced eculizumab was within the equivalence margin of 80.00% to 125.00%, respectively. Other objectives for the study were to evaluate safety, tolerability, immunogenicity, and PD profiles for the IPs.Results: A total of 240 subjects (80 in each treatment group) were enrolled. Back transformation provided the geometric LSMean ratio for the comparison of SB12/EU sourced eculizumab, SB12/US sourced eculizumab and EU sourced eculizumab/US sourced eculizumab for AUC inf were 99.1 % (95.41,102.85), 95.1 % (91.40, 99.04), and 96.0 % (92.16, 100.10), respectively. The corresponding 90% CI was within the pre-defined equivalence margin of 80.00-125.00%, indicating that the each of two treatments are bioequivalent.The profiles of mean terminal complement activity and mean change from baseline of complement activity were superimposable following administration of SB12, EU sourced eculizumab, and US sourced eculizumab. There was a rapid decrease in the complement activity at the end of infusion and then a slow restoration. There was no non-responder in the aspect of the measured complement activity after treatment.There were no deaths or discontinuation of IP due to treatment-emergent adverse events (TEAEs) during the study. Two serious adverse events (SAEs) (renal colic in the SB12 treatment group and back pain in the US eculizumab treatment group) were reported, in 2 subjects. Both events were considered not related to the IP. The proportion of subjects who experienced TEAEs were similar between the SB12, EU sourced eculizumab, and US sourced eculizumab treatment groups (70.0%, 65.0%, and 71.3% of subjects, respectively). The overall incidence of subjects with post-dose anti-drug antibodies (ADA) to eculizumab was 2 (2.5%), 1 (1.3%), and 0 (0.0%) subjects in the SB12, EU sourced eculizumab, and US sourced eculizumab treatment groups, respectively. There was no significant difference between treatment groups. None of the subjects with post-dose ADA to eculizumab had a positive result for neutralizing antibodies.Conclusion: The Phase I study demonstrated PK bioequivalence and showed comparable PD, safety, immunogenicity between SB12 and the RP eculizumab. DisclosuresLee: Samsung Bioepis, Co., Ltd.: Current Employment. Jang: Samsung Bioepis, Co., Ltd.: Current Employment. Kim: Samsung Bioepis, Co., Ltd.: Current Employment. Jeong: Samsung Bioepis, Co., Ltd.: Current Employment. Lee: Samsung Bioepis, Co., Ltd.: Current Employment. Jung: Samsung Bioepis, Co., Ltd.: Current Employment. Demichelis: Novartis: Consultancy, Research Funding, Speakers Bureau; Bristol/Celgene: Consultancy, Speakers Bureau; AMGEN: Consultancy, Speakers Bureau; ASH: Research Funding; Jazz: Consultancy; Gilead: Consultancy; Astellas: Consultancy; Abbvie: Consultancy, Speakers Bureau.

Human Complement C4B Allotypes and Deficiencies in Selected Cases With Autoimmune Diseases

Human complement C4 is one of the most diverse but heritable effectors for humoral immunity. To help understand the roles of C4 in the defense and pathogenesis of autoimmune and inflammatory diseases, we determined the bases of polymorphisms including the frequent genetic deficiency of C4A and/or C4B isotypes. We demonstrated the diversities of C4A and C4B proteins and their gene copy number variations (CNVs) in healthy subjects and patients with autoimmune disease, such as type 1 diabetes, systemic lupus erythematosus (SLE) and encephalitis. We identified subjects with (a) the fastest migrating C4B allotype, B7, or (b) a deficiency of C4B protein caused by genetic mutation in addition to gene copy-number variation. Those variants and mutants were characterized, sequenced and specific techniques for detection developed. Novel findings were made in four case series. First, the amino acid sequence determinant for C4B7 was likely the R729Q variation at the anaphylatoxin-like region. Second, in healthy White subject MS630, a C-nucleotide deletion at codon-755 led to frameshift mutations in his single C4B gene, which was a private mutation. Third, in European family E94 with multiplex lupus-related mortality and low serum C4 levels, the culprit was a recurrent haplotype with HLA-A30, B18 and DR7 that segregated with two defective C4B genes and identical mutations at the donor splice site of intron-28. Fourth, in East-Asian subject E133P with anti-NMDA receptor encephalitis, the C4B gene had a mutation that changed tryptophan-660 to a stop-codon (W660x), which was present in a haplotype with HLA-DRB1*04:06 and B*15:27. The W660x mutation is recurrent among East-Asians with a frequency of 1.5% but not detectable among patients with SLE. A meticulous annotation of C4 sequences revealed clusters of variations proximal to sites for protein processing, activation and inactivation, and binding of interacting molecules.

The Protease SplB of Staphylococcus aureus Targets Host Complement Components and Inhibits Complement-Mediated Bacterial Opsonophagocytosis.

ABSTRACTStaphylococcus aureus is an opportunistic pathogen that can cause life-threatening infections, particularly in immunocompromised individuals. The high-level virulence of S. aureus largely relies on its diverse and variable collection of virulence factors and immune evasion proteins, including the six serine protease-like proteins SplA to SplF. Spl proteins are expressed by most clinical isolates of S. aureus, but little is known about the molecular mechanisms by which these proteins modify the host’s immune response for the benefit of the bacteria. Here, we identify SplB as a protease that inactivates central human complement proteins, i.e., C3, C4, and the activation fragments C3b and C4b, by preferentially cleaving their α-chains. SplB maintained its proteolytic activity in human serum, degrading C3 and C4. SplB further cleaved the components of the terminal complement pathway, C5, C6, C7, C8, and C9. In contrast, the important soluble human complement regulators factor H and C4b-binding protein (C4BP), as well as C1q, were left intact. Thereby, SplB reduced C3b-mediated opsonophagocytosis by human neutrophils as well as C5b-9 deposition on the bacterial surface. In conclusion, we identified the first physiological substrates of the S. aureus extracellular protease SplB. This enzyme inhibits all three complement pathways and blocks opsonophagocytosis. Thus, SplB can be considered a novel staphylococcal complement evasion protein.IMPORTANCE The success of bacterial pathogens in immunocompetent humans depends on the control and inactivation of host immunity. S. aureus, like many other pathogens, efficiently blocks host complement attack early in infection. Aiming to understand the role of the S. aureus-encoded orphan proteases of the Spl operon, we asked whether these proteins play a role in immune escape. We found that SplB inhibits all three complement activation pathways as well as the lytic terminal complement pathway. This blocks the opsonophagocytosis of the bacteria by neutrophils. We also clarified the molecular mechanisms: SplB cleaves the human complement proteins C3, C4, C5, C6, C7, C8, and C9 as well as factor B but not the complement inhibitors factor H and C4BP. Thus, we identify the first physiological substrates of the extracellular protease SplB of S. aureus and characterize SplB as a novel staphylococcal complement evasion protein.

SVCT2 Overexpression and Ascorbic Acid Uptake Increase Cortical Neuron Differentiation, Which Is Dependent on Vitamin C Recycling between Neurons and Astrocytes.

During brain development, sodium–vitamin C transporter (SVCT2) has been detected primarily in radial glial cells in situ, with low-to-absent expression in cerebral cortex neuroblasts. However, strong SVCT2 expression is observed during the first postnatal days, resulting in increased intracellular concentration of vitamin C. Hippocampal neurons isolated from SVCT2 knockout mice showed shorter neurites and low clustering of glutamate receptors. Other studies have shown that vitamin C-deprived guinea pigs have reduced spatial memory, suggesting that ascorbic acid (AA) and SVCT2 have important roles in postnatal neuronal differentiation and neurite formation. In this study, SVCT2 lentiviral overexpression induced branching and increased synaptic proteins expression in primary cultures of cortical neurons. Analysis in neuroblastoma 2a (Neuro2a) and human subventricular tumor C3 (HSVT-C3) cells showed similar branching results. SVCT2 was mainly observed in the cell membrane and endoplasmic reticulum; however, it was not detected in the mitochondria. Cellular branching in neuronal cells and in a previously standardized neurosphere assay is dependent on the recycling of vitamin C or reduction in dehydroascorbic acid (DHA, produced by neurons) by glial cells. The effect of WZB117, a selective glucose/DHA transporter 1 (GLUT1) inhibitor expressed in glial cells, was also studied. By inhibiting GLUT1 glial cells, a loss of branching is observed in vitro, which is reproduced in the cerebral cortex in situ. We concluded that vitamin C recycling between neurons and astrocyte-like cells is fundamental to maintain neuronal differentiation in vitro and in vivo. The recycling activity begins at the cerebral postnatal cortex when neurons increase SVCT2 expression and concomitantly, GLUT1 is expressed in glial cells.

MiR-92d-3p suppresses the progression of diabetic nephropathy renal fibrosis by inhibiting the C3/HMGB1/TGF-β1 pathway.

The pathogenesis of diabetic nephropathy (DN) has not been fully elucidated. MicroRNAs (miRNAs) play an important role in the onset and development of DN renal fibrosis. Thus, the present study aimed to investigate the effect of miR-92d-3p on the progression of DN renal fibrosis. We used qRT-PCR to detect the expression levels of miR-92d-3p in the kidneys of patients with DN. Then, after transfecting lentiviruses containing miR-92d-3p into the kidneys of a DN mouse model and HK-2 cell line, we used qRT-PCR to detect the expression levels of miR-92d-3p, C3, HMGB1, TGF-β1, α-SMA, E-cadherin, and Col I. The expression levels of interleukin (IL) 1β (IL-1β), IL-6, and tumor necrosis factor-α (TNF-α) in the HK-2 cells were detected through enzyme-linked immunosorbent assay (ELISA), and Western blotting and immunofluorescence were used in detecting the expression levels of fibronectin, α-SMA, E-cadherin, and vimentin. Results showed that the expression levels of miR-92d-3p in the kidney tissues of patients with DN and DN animal model mice decreased, and C3 stimulated HK-2 cells to produce inflammatory cytokines. The C3/HMGB1/TGF-β1 pathway was activated, and epithelial-to-interstitial transition (EMT) was induced in the HK-2 cells after human recombinant C3 and TGF-β1 protein were added. miR-92d-3p inhibited inflammatory factor production by C3 in the HK-2 cells and the activation of the C3/HMGB1/TGF-β1 pathway and EMT by C3 and TGF-β1. miR-92d-3p suppressed the progression of DN renal fibrosis by inhibiting the activation of the C3/HMGB1/TGF-β1 pathway and EMT.

AVB-500, a selective inhibitor of GAS6-AXL, shows improved therapeutic efficacy in combination with paclitaxel in uterine serous carcinoma

<h3>Objectives:</h3> The receptor tyrosine kinase AXL is highly expressed in uterine serous cancer (USC) and has been found to mediate chemoresistance. Recently, in a Phase I clinical trial in ovarian cancer, AVB-500 has been shown in combination with paclitaxel to be safe and tolerable. The objectives of this study were to determine if AVB-500 improves response to paclitaxel in USC in preclinical studies and to explore how GAS6/AXL expression modulates the immune environment via changes in cytokine secretion profiling. <h3>Methods:</h3> AVB-500 (AVB) was supplied by Aravive Biologics. XTT cell viability was performed with two USC tumor cells - ARK1 and primary cells derived from ascites of a patient with platinum refractory USC (PUC1). Cells were treated with paclitaxel (P) alone and with a combination of AVB-500 and paclitaxel (AVB+P). Western blot analysis for apoptosis marker, cleaved PARP (c-PARP) was completed with ARK1 cells treated with AVB, P, and AVB+P. <i>in vivo</i> studies were performed using NOD-SCID mice injected with 1 x 107 ARK1 cells intraperionteally (IP). Treatment groups included vehicle, AVB, P, and AVB+P. Tumor nodule number, volume, and weight were assessed for each treatment group. Profiling of 80 inflammatory cytokines was performed with conditioned media (CM) collected from ARK1 control and AXL knockdown (KD) cells using Human Cytokine Array C5 (RayBiotech). Per manufacturer's criteria, proteins were significantly upregulated if the signal intensity ≥1.50 fold, and significantly downregulated if the signal intensity ≤0.65 fold. <h3>Results:</h3> We found that the USC cell line ARK1 had decreased viability when treated with A+P than when treated with P alone (IC50 11.5nM vs 39.2nM). In addition, the relative percent viability was lower in the AVB+P treated cells for every concentration of P (0.156 nM to 320 nM, p<0.05). Similarly, PUC1 had decreased viability when treated with AVB+P than with P alone (IC50 20.9nM vs 43.3nM). Western blot revealed greater apoptosis in the AVP+P treated cells as seen by c-PARP expression when compared to P alone. An <i>in vivo</i> metastatic IP model had significantly fewer tumors when treated with AVB+P compared to P alone with number of tumor nodules ≤1mm (2.750 vs 7.889, p=0.028) as well as decreased tumor weight (0.025 g vs 0.079 g, p<0.001) and smaller tumor volume (16.75 mm3 vs 72.33 mm3, p=0.002). Mouse weights were similar between treatment groups, indicating a favorable safety profile of the AVB+P combination. Furthermore, the CM from ARK1 cells with and without AXL expression were compared and the following significant changes were identified in the AXL non-expressing CM: decrease in cytokines involved in tumor cell proliferation and survival (TNF<b>-</b>α TNF-β<b>,</b> IGF, HGF, and TGF<b>-</b>β3), decrease in pro-tumor cytokines (IL-10, IL-13, and IL-16), and a decrease in angiogenesis factors (angiogenin, PDGF, FGF-6, and FGF-7). <h3>Conclusions:</h3> The combination of AVB-500 and paclitaxel has demonstrates a superior therapeutic efficacy compared to paclitaxel alone in USC pre-clinical models. In addition, cytokine expression profiles for tumor cell proliferation, pro-tumor cytokines, and angiogenesis factors suggest a mechanism by which GAS6/AXL signaling leads to immune suppression and evasion. Experiments in additional USC cell lines and patient-derived xenograft models are ongoing.

Analytical Similarity Assessment of ABP 959 in Comparison with Eculizumab Reference Product.

BackgroundABP 959 is one of the first proposed biosimilars to eculizumab reference product (RP), a recombinant IgG2/4Ƙ monoclonal antibody (mAb) that binds human C5 complement protein and inhibits C5 cleavage to C5a and C5b, preventing the generation of the terminal complement complex C5b-9. Eculizumab RP is approved for the treatment of paroxysmal nocturnal hemoglobinuria, atypical hemolytic uremic syndrome, myasthenia gravis in patients who are anti-acetylcholine receptor antibody positive, and neuromyelitis optica spectrum disorder in patients who are anti-aquaporin-4 antibody positive.ObjectivesThe objective of this work was to comparatively assess analytical (structural and functional) similarity between ABP 959 and eculizumab RP using sensitive, state-of-the art analytical methods capable of detecting minor differences in product quality attributes.MethodsComprehensive analytical (structural and functional) characterization utilizing orthogonal techniques was performed using multiple lots of ABP 959 and eculizumab RP over several years applying > 40 state-of-the-art assays. Comparisons were performed to investigate the primary structure and post-translational modifications including glycans, higher-order structure, particles and aggregates, product-related structures and impurities, thermal stability and forced degradation, general properties, and biological properties mediated by target binding.ResultsResults confirmed that ABP 959 had the same amino acid sequence, similar primary structure, higher-order structure, post-translational profiles, and the same protein content and concentration (e.g., ABP 959: 9.4–10.0; eculizumab EU: 9.4–10.0; eculizumab US: 9.3–10.3 mg/mL) as well as biological activity as eculizumab RP.ConclusionsBased on these results, it can be concluded that ABP 959 is analytically similar to eculizumab RP.

Clinical Anatomy of Human Donor C7 Nerve Roots for Surgical Transfer in Patients with Spastic Arm Paralysis

Contralateral C7 (CC7) nerve transfer has successfully restored hand function in patients with spastic hemiplegia from chronic central nervous system injuries. However, little is known about the morphology and anatomy of the donor C7 nerve root in patients undergoing this procedure. This study quantified intraoperative measurements of donor C7 nerve roots during CC7 transfer surgery for spastic hemiplegia in patients treated at a high-volume center to describe observed anatomical variations for successful direct anastomosis. A database of images from 21 patients (2 females, 19 males) undergoing CC7 surgery was searched for photographic data that contained a standard ruler measuring donor C7 nerve root length after surgical sectioning and before transfer. Two independent observers analyzed these images and recorded C7 nerve root diameter, length, and branch lengths. Mean (SD) values of donor C7 nerve measurements were length, 53.5 (8.0) mm; diameter, 5.1 (0.9) mm; branch length following surgical sectioning, 18.3 (6.3) mm. Right-sided donor C7 nerve roots yielded significantly longer branches compared with left-sided donor C7 nerve roots (P= 0.01). Other patient factors such as age, sex, or laterality of brain injury did not influence intraoperative anatomy. We report detailed intraoperative measurements of the donor C7 root during CC7 nerve transfer for spastic hemiplegia. These findings describe existing variation in surgical C7 nerve root anatomy in patients undergoing this procedure and may serve as a general reference for the expected donor C7 length in successful direct anastomosis.

Evaluation of Antimicrobial Activity of the C3f Peptide, a Derivative of Human C3 Protein

Evaluation of Antimicrobial Activity of the C3f Peptide, a Derivative of Human C3 Protein

Enhanced detection of expanded repeat mRNA foci with hybridization chain reaction

Transcribed nucleotide repeat expansions form detectable RNA foci in patient cells that contribute to disease pathogenesis. The most widely used method for detecting RNA foci, fluorescence in situ hybridization (FISH), is powerful but can suffer from issues related to signal above background. Here we developed a repeat-specific form of hybridization chain reaction (R-HCR) as an alternative method for detection of repeat RNA foci in two neurodegenerative disorders: C9orf72 associated ALS and frontotemporal dementia (C9 ALS/FTD) and Fragile X-associated tremor/ataxia syndrome. R-HCR to both G4C2 and CGG repeats exhibited comparable specificity but > 40 × sensitivity compared to FISH, with better detection of both nuclear and cytoplasmic foci in human C9 ALS/FTD fibroblasts, patient iPSC derived neurons, and patient brain samples. Using R-HCR, we observed that integrated stress response (ISR) activation significantly increased the number of endogenous G4C2 repeat RNA foci and triggered their selective nuclear accumulation without evidence of stress granule co-localization in patient fibroblasts and patient derived neurons. These data suggest that R-HCR can be a useful tool for tracking the behavior of repeat expansion mRNA in C9 ALS/FTD and other repeat expansion disorders.

Infection of Bronchial Epithelial Cells by the Human Adenoviruses A12, B3, and C2 Differently Regulates the Innate Antiviral Effector APOBEC3B.

Human adenoviruses (HAdVs) are a large family of DNA viruses that include more than 100 genotypes divided into seven species (A to G) and induce respiratory tract infections, gastroenteritis, and conjunctivitis. Genetically modified adenoviruses are also used as vaccines, gene therapies, and anticancer treatments. The APOBEC3s are a family of cytidine deaminases that restrict viruses by introducing mutations in their genomes. Viruses developed different strategies to cope with the APOBEC3 selection pressure, but nothing is known on the interplay between the APOBEC3s and the HAdVs. In this study, we focused on three HAdV strains: the B3 and C2 strains, as they are very frequent, and the A12 strain, which is less common but is oncogenic in animal models. We demonstrated that the three HAdV strains induce a similar APOBEC3B upregulation at the transcriptional level. At the protein level, however, APOBEC3B is abundantly expressed during HAdV-A12 and -C2 infection and shows a nuclear distribution. On the contrary, APOBEC3B is barely detectable in HAdV-B3-infected cells. APOBEC3B deaminase activity is detected in total protein extracts upon HAdV-A12 and -C2 infection. Bioinformatic analysis demonstrates that the HAdV-A12 genome bears a stronger APOBEC3 evolutionary footprint than that of the HAdV-C2 and HAdV-B3 genomes. Our results show that HAdV infection triggers the transcriptional upregulation of the antiviral innate effector APOBEC3B. The discrepancies between the APOBEC3B mRNA and protein levels might reflect the ability of some HAdV strains to antagonize the APOBEC3B protein. These findings point toward an involvement of APOBEC3B in HAdV restriction and evolution. IMPORTANCE The APOBEC3 family of cytosine deaminases has important roles in antiviral innate immunity and cancer. Notably, APOBEC3A and APOBEC3B are actively upregulated by several DNA tumor viruses and contribute to transformation by introducing mutations in the cellular genome. Human adenoviruses (HAdVs) are a large family of DNA viruses that cause generally asymptomatic infections in immunocompetent adults. HAdVs encode several oncogenes, and some HAdV strains, like HAdV-A12, induce tumors in hamsters and mice. Here, we show that HAdV infection specifically promotes the expression of the APOBEC3B gene. We report that infection with the A12 strain induces a strong expression of an enzymatically active APOBEC3B protein in bronchial epithelial cells. We provide bioinformatic evidence that HAdVs' genomes and notably the A12 genome are under APOBEC3 selection pressure. Thus, APOBEC3B might contribute to adenoviral restriction, diversification, and oncogenic potential of particular strains.

Recombinant human C1 esterase inhibitor for hereditary angioedema attacks: A European registry

BackgroundHereditary angioedema (HAE) due to C1 esterase inhibitor deficiency (C1-INH-HAE) is characterized by recurrent swelling attacks. A European treatment registry was established to review the adverse event profile and efficacy of recombinant human C1 esterase inhibitor (rhC1-INH) for HAE attacks. MethodsIndividuals with C1-INH-HAE were enrolled following a decision to treat with rhC1-INH and provision of written informed consent. Medical history and baseline HAE information were collected at screening. Healthcare providers entered data on HAE attacks, response to treatment, and adverse events using a web-based questionnaire. ResultsFrom July 1, 2011, through December 1, 2019, 71 patients with C1-INH-HAE (30 male/41 female; mean age, 47.3 years; age range, 19–78 years) in 9 countries reported 2356 attacks and were treated with rhC1-INH. Before registry entry, patients, including 20 (28.2%) who were on maintenance therapy/prophylaxis at registry enrollment, experienced a mean of 25 HAE attacks per year (median, 16 [range, 0–185]). Most treated HAE attacks were abdominal (46.1%), followed by peripheral (38.3%), oro-facial-pharyngeal (14.8%), urogenital (3.2%), and laryngeal (2.6%). The mean rhC1-INH dose was 3307 U (43.3 U/kg). Patients reported symptom improvement within 4 h for 97.8% of attacks (2305/2356) with rhC1-INH; most attacks (99.8%; 2351/2356) required only 1 dose. Five attacks were treated with a second dose (total rhC1-INH dose administered for attack, 4200 U). No hypersensitivity, thrombotic/thromboembolic events, or drug-related serious adverse events were reported. ConclusionThe rhC1-INH treatment registry provided real-world data on the treatment of 2356 HAE attacks that were consistent with clinical trial data of rhC1-INH in patients with C1-INH-HAE.

Correlation between different instrumentation variants and the degree of destabilization in treating cervical spondylotic spinal canal stenosis by unilateral hemilaminectomy with bilateral decompression: a biomechanical investigation

PurposeUnilateral hemilaminectomy with bilateral decompression (BDZ) was proposed as an alternative decompressive procedure in cervical spondylotic myelopathy (CSM). Despite promising clinical results, the destabilizing effect is yet unknown. We therefore performed a biomechanical study to investigate whether lateral mass screw fixation should follow BDZ.MethodsSix human C2–C7 cervical specimens were tested under various conditions: native, unilateral hemilaminectomy with bilateral decompression without/with fixation (BDZ/BDF), unilateral hemilaminectomy with bilateral decompression and unilateral foraminotomy without/with fixation (UFZ/UFF), unilateral hemilaminectomy with bilateral decompression and bilateral foraminotomy without/with fixation (BFZ/BFF), and laminectomy without/with fixation (LAZ/LAF). Instrumention was applied from C3–C6. For each condition, the three-dimensional kinematics of the cervical specimen were measured in three main loading directions with an ultrasonic motion analysis system. ANOVA was used to determine differences between the specific segment conditions to assess the parameter’s range of motion (ROM) and neutral zone (NZ).ResultsFor flexion–extension, lateral bending and axial rotation, ROM of BDZ, UFZ, BFZ and LAZ remained at the level of the native condition (p > 0.74), whereas fixation reduced ROM significantly (p < 0.01). Between BDF, UFF, BFF and LAF, no significant differences in reduction in ROM were seen (p > 0.49). Results for NZ were equivalent to ROM in flexion–extension and lateral bending. For axial rotation, NZ remained almost constant on the native level for all tested conditions.ConclusionBilateral decompression via a hemilaminectomy, even if combined with foraminotomy, could be a less invasive treatment option for multilevel CSM in patients with lordotic cervical alignment and absence of segmental instability.

Identification of filovirus entry inhibitors targeting the endosomal receptor NPC1 binding site

Filoviruses, mainly consisting of Ebola viruses (EBOV) and Marburg viruses (MARV), are enveloped negative-strand RNA viruses which can infect humans to cause severe hemorrhagic fevers and outbreaks with high mortality rates. The filovirus infection is mediated by the interaction of viral envelope glycoprotein (GP) and the human endosomal receptor Niemann-Pick C1 (NPC1). Blocking this interaction will prevent the infection. Therefore, we utilized an In silico screening approach to conduct virtual compound screening against the NPC1 receptor-binding site (RBS). Twenty-six top-hit compounds were purchased and evaluated by in vitro cell based inhibition assays against pseudotyped or replication-competent filoviruses. Two classes (A and U) of compounds were identified to have potent inhibitory activity against both Ebola and Marburg viruses. The IC50 values are in the lower level of micromolar concentrations. One compound (compd-A) was found to have a sub-micromolar IC50 value (0.86 μM) against pseudotyped Marburg virus. The cytotoxicity assay (MTT) indicates that compd-A has a moderate cytotoxicity level but the compd-U has much less toxicity and the CC50 value was about 100 μM. Structure-activity relationship (SAR) study has found some analogs of compd-A and -U have reduced the toxicity and enhanced the inhibitory activity. In conclusion, this work has identified several qualified lead-compounds for further drug development against filovirus infection.

Population pharmacokinetics of recombinant human C1 esterase inhibitor in children with hereditary angioedema

BackgroundRecombinant human C1 esterase inhibitor (rhC1-INH) is indicated in the United States for the treatment of acute hereditary angioedema (HAE) attacks in adolescents and adults; it is also indicated in Europe for children aged 2 years and older. A need exists for further insight into potential pharmacokinetic (PK) differences in functional C1-INH levels by age (ie, children, adolescents, and adults). ObjectiveTo perform population PK modeling to predict C1-INH levels by age after by age rhC1-INH administration. MethodsData from a phase 2 pediatric trial (children aged 4-13 years at screening) were added to a database of 6 trials in adults and adolescents. An unpublished population PK model was refined and used to simulate C1-INH exposure. ResultsAnalysis included 153 individuals (14 healthy volunteers; 139 patients with HAE) and 1788 functional C1-INH measurements (59 from 20 patients in the pediatric trial). Bodyweight (population weight, 16-128 kg) was a key predictor of C1-INH volume of distribution. Age was not a predictor of C1-INH PK after the inclusion of bodyweight in the model. Simulations of the recommended rhC1-INH dosing regimen (bodyweight <84 kg, 50 U/kg; ≥84 kg, 4200 U) revealed that overall C1-INH exposure was comparable among age groups. Predicted peak functional C1-INH concentrations were at or above the lower level of normal (≥0.7 U/mL) for 99.8% of adults (≥18 years), 99.8% of adolescents (14-17 years), and 96.0% of children (2-13 years). ConclusionThe analyses support the same weight-based rhC1-INH dosing for HAE attacks in children as currently recommended for adolescents and adults. These results support clinical trial data, which revealed similar safety and efficacy profiles across these age groups.

Treatment of Hereditary Angioedema.

Hereditary angioedema due to C1-esterase inhibitor deficiency (C1-INH-HAE) is a rare autosomal dominant disease. In the last decade, new drugs and new indications for old drugs have played a role in the management of C1-INH-HAE. This review examines current therapy for C1-INH-HAE and provides a brief summary of drugs that are under development. Increased knowledge of the pathophysiology of C1-INH-HAE has been crucial for advances in the field, with inhibition of the kallikrein-kinin system (plasma kallikrein, activated factor XII) as a key area in the discovery of new drugs, some of which are already marketed for treatment of C1-INH-HAE. Pharmacological treatment is based on 3 pillars: treatment of acute angioedema attacks (on-demand treatment), short-term (preprocedure) prophylaxis, and long-term prophylaxis. The 4 drugs that are currently available for the treatment of acute angioedema attacks (purified plasma-derived human C1 esterase inhibitor concentrate, icatibant acetate, ecallantide, recombinant human C1 esterase inhibitor) are all authorized for self-administration, except ecallantide. Purified plasma-derived human C1 esterase inhibitor concentrate is the treatment of choice for short-term prophylaxis. Tranexamic acid, danazol, intravenous and subcutaneous nanofiltered purified plasma-derived human C1 esterase inhibitor concentrate, and lanadelumab can be used for long-term prophylaxis. New drugs are being investigated, mainly as long-term prophylaxis, and are aimed at blocking the kallikrein-kinin system by means of antiprekallikrein, antikallikrein, and anti-activated FXII action.

Purification of Human Complement Component C4 and Sample Preparation for Structural Biology Applications.

Activated complement component C4 (C4b) is the nonenzymatic component of the classical pathway (CP) convertases of the complement system. Preparation of C4 and C4b samples suitable for structural biology studies is challenging due to low yields and complexity of recombinant C4 production protocols reported so far and heterogeneity of C4 in native sources. Here we present a purification protocol for human C4 and describe sample preparation methods for structural investigation of C4 and its complexes by crystallography, small angle X-ray scattering, and electron microscopy.

Cultured Cell Line Models of Neuronal Differentiation: NT2, PC12, and SK-N-MC.

The lack of a convenient, easily maintained, and inexpensive in vitro human neuronal model to study neurodegenerative diseases prompted us to develop a rapid, 1-h differentiated neuronal cell model based on human NT2 cells and C3 transferase. Here, we describe the rapid differentiation of human neuronal NT2 cells, and the differentiation, transduction, and transfection of human SK-N-MC cells and rat PC12 cells to obtain cells with the morphology of differentiated neurons that can express exogenous genes of interest at high level.

Therapeutic Efficacy of a Trichinella Spiralis Paramyosin-Derived Peptide Modified With a Membrane-Targeting Signal in Mice With Antigen-Induced Arthritis.

Helminth-derived molecules have the ability to modulate the host immune system. Our previous study identified a tetradecapeptide derived from Trichinella spiralis paramyosin (Ts-pmy) that could bind to human complement component C9 to inhibit its polymerization, making the peptide a candidate therapeutic agent for complement-related immune disorders. Here, the peptide underwent an N-terminal modification with a membrane-targeting signal (a unique myristoylated peptide) to improve its therapeutic efficacy. We found that the modified peptide had a binding affinity to human C9 that was similar to that of the original peptide, as confirmed by microscale thermophoresis assays. The binding of the modified peptide to human C9 resulted in the inhibition of C9-related complement activation, as reflected by the decreased Zn2+-induced C9 polymerization and the decreased C9-dependent lysis of rabbit erythrocytes. In addition, the original and modified peptides could both bind to recombinant mouse C9 and inhibit the C9-dependent lysis of rabbit erythrocytes in normal mouse serum (NMS), which meant that the peptides could cross the species barrier to inhibit complement activity in mice. Further in vitro and in vivo analyses confirmed that the peptide modification increased the retention time of the peptide. Furthermore, intraarticular injection of the modified peptide markedly ameliorated knee swelling and joint damage in mice with antigen-induced arthritis (AIA), as assessed histologically. These results suggested that the Ts-pmy-derived peptide modified with a membrane-targeting signal was a reasonable candidate therapeutic agent for membrane attack complex (MAC)-related diseases [such as rheumatoid arthritis (RA)] and the study presented a new modification method to improve the potential therapeutic effects of the peptide.