Activation of the alternative complement pathway modulates inflammation in thoracic aortic aneurysm/dissection.

Thoracic aortic aneurysm/dissection (TAAD) is a lethal vascular disease, and several pathological factors participate in aortic medial degeneration. We previously discovered that the complement C3a-C3aR axis in smooth muscle cells promotes the development of thoracic aortic dissection (TAD) through regulation of matrix metalloproteinase 2. However, discerning the specific complement pathway that is activated and elucidating how inflammation of the aortic wall is initiated remain unknown. We ascertained that the plasma levels of C3a and C5a were significantly elevated in patients with TAD and that the levels of C3a, C4a, and C5a were higher in acute TAD than in chronic TAD. We also confirmed the activation of the complement in a TAD mouse model. Subsequently, knocking out Cfb (Cfb) or C4 in mice with TAD revealed that the alternative pathway and Cfb played a significant role in the TAD process. Activation of the alternative pathway led to generation of the anaphylatoxins C3a and C5a, and knocking out their receptors reduced the recruitment of inflammatory cells to the aortic wall. Moreover, we used serum from wild-type mice or recombinant mice Cfb as an exogenous source of Cfb to treat Cfb KO mice and observed that it exacerbated the onset and rupture of TAD. Finally, we knocked out Cfb in the FBN1C1041G/+ Marfan-syndrome mice and showed that the occurrence of TAA was reduced. In summary, the alternative complement pathway promoted the development of TAAD by recruiting infiltrating inflammatory cells. Targeting the alternative pathway may thus constitute a strategy for preventing the development of TAAD.NEW & NOTEWORTHY The alternative complement pathway promoted the development of TAAD by recruiting infiltrating inflammatory cells. Targeting the alternative pathway may thus constitute a strategy for preventing the development of TAAD.

Expansion of Anticomplement Therapy Indications from Rare Genetic Disorders to Common Kidney Diseases.

Complement constitutes a major part of the innate immune system. The study of complement in human health has historically focused on infection risks associated with complement protein deficiencies; however, recent interest in the field has focused on overactivation of complement as a cause of immune injury and the development of anticomplement therapies to treat human diseases. The kidneys are particularly sensitive to complement injury, and anticomplement therapies for several kidney diseases have been investigated. Overactivation of complement can result from loss-of-function mutations in complement regulators; gain-of-function mutations in key complement proteins such as C3 and factor B; or autoantibody production, infection, or tissue stresses, such as ischemia and reperfusion, that perturb the balance of complement activation and regulation. Here, we provide a high-level review of the status of anticomplement therapies, with an emphasis on the transition from rare diseases to more common kidney diseases.

MASP3 Deficiency in Mice Reduces but Does Not Abrogate Alternative Pathway Complement Activity Due to Intrinsic Profactor D Activity.

Complement factor D (FD) is a rate-limiting enzyme of the alternative pathway (AP). Recent studies have suggested that it is synthesized as an inactive precursor and that its conversion to enzymatically active FD is catalyzed by mannan-binding lectin-associated serine protease 3 (MASP3). However, whether MASP3 is essential for AP complement activity remains uncertain. It has been shown that Masp1/3 gene knockout did not prevent AP complement overactivation in a factor H-knockout mouse, and a human patient lacking MASP3 still retained AP complement activity. In this study, we have assessed AP complement activity in a Masp3-knockout mouse generated by CRISPR/Cas9 editing of the Masp1/3 gene. We confirmed specific Masp3 gene inactivation by showing intact MASP1 protein expression and absence of mature FD in the mutant mice. Using several assays, including LPS- and zymosan-induced C3b deposition and rabbit RBC lysis tests, we detected plasma concentration-dependent AP complement activity in Masp3 gene-inactivated mice. Thus, although not measurable in 5% plasma, significant AP complement activity was detected in 20-50% plasma of Masp3 gene-inactivated mice. Furthermore, whereas FD gene deletion provided more than 90% protection of CD55/Crry-deficient RBCs from AP complement-mediated extravascular hemolysis, Masp3 gene deletion only provided 30% protection in the same study. We also found pro-FD to possess intrinsic catalytic activity, albeit at a much lower level than mature FD. Our data suggest that MASP3 deficiency reduces but does not abrogate AP complement activity and that this is explained by intrinsic pro-FD activity, which can be physiologically relevant in vivo.

Neutrophil-activating therapy for the treatment of cancer.

Despite their cytotoxic capacity, neutrophils are often co-opted by cancers to promote immunosuppression, tumor growth, and metastasis. Consequently, these cells have received little attention as potential cancer immunotherapeutic agents. Here, we demonstrate in mouse models that neutrophils can be harnessed to induce eradication of tumors and reduce metastatic seeding through the combined actions of tumor necrosis factor, CD40 agonist, and tumor-binding antibody. The same combination activates human neutrophils in vitro, enabling their lysis of human tumor cells. Mechanistically, this therapy induces rapid mobilization and tumor infiltration of neutrophils along with complement activation in tumors. Complement component C5a activates neutrophils to produce leukotriene B4, which stimulates reactive oxygen species production via xanthine oxidase, resulting in oxidative damage and T cell-independent clearance of multiple tumor types. These data establish neutrophils as potent anti-tumor immune mediators and define an inflammatory pathway that can be harnessed to drive neutrophil-mediated eradication of cancer.

Mapping the T cell epitopes of the M-type transmembrane phospholipase A2 receptor in primary membranous nephropathy.

The M-type phospholipase A2 receptor (PLA2R) is the major autoantigen of primary membranous nephropathy (MN). Despite many studies on B-cell epitopes recognized by antibodies, little is known about T-cell epitopes. Herein, we synthesized 123 linear peptides, each consisting of 15-22 amino acids with 8-12 amino acid overlaps, across ten domains of PLA2R. Their binding capacity to risk (DRB1∗1501, DRB1∗0301) and protective (DRB1∗0901, DRB1∗0701) HLA molecules was then assessed by flow cytometry. Proliferation of CD4+ T cells from patients with anti-PLA2R positive MN was analyzed after peptide stimulation. Cytokines produced by activated peripheral blood mononuclear cells were measured by cytometric bead arrays. We identified 17 PLA2R peptides that bound to both DRB1∗1501 and DRB1∗0301 molecules with high capacity. Some of these peptides showed decreased binding to heterozygous DRB1∗1501/0901 and DRB1∗0301/0701. Ten of the 17 peptides (CysR1, CysR10, CysR12, FnII-3, CTLD3-9, CTLD3-10, CTLD3-11, CTLD5-2-1, CTLD7-1 and CTLD7-2) induced significant proliferation of CD4+ T cells from patients with MN than cells from healthy individuals. Upon activation by these peptides, peripheral blood mononuclear cells from patients with MN produced higher levels of pro-inflammatory cytokines, predominantly IL-6, TNF-α, IL-10, IL-9 and IL-17. Thus, we mapped and identified ten peptides in the CysR, FnII, CTLD3, CTLD5, and CTLD7 domains of PLA2R as potential T-cell epitopes of MN. These findings are a first step towards developing peptide-specific immunotherapies.

A disturbed balance between blood complement protective factors (FH, ApoE) and common pathway effectors (C5a, TCC) in acute COVID-19 and during convalesce.

A complement effect on homeostasis during infection is determined by both cytotoxic (activate complement component 5 (C5a) terminal cytotoxic complex (TCC)), and cytoprotective elements (complement factor H (FH), as well as apolipoprotein E (ApoE)). Here, we investigated the gap in knowledge in their blood milieu during SARS-CoV-2 infection with respect to the viral burden, level of tissue necrosis, and immunological response. 101 patients hospitalized with a PCR-confirmed diagnosis of COVID-19 had blood collected at H1 (48 h), H2 (3-4 Days), H3 (5-7 days), H4 (more than 7 days up to 93 days). Pre-existing conditions, treatment, the incidence of cerebrovascular events (CVA), a history of deep venous thrombosis (DVT) and pulmonary embolism (PE), and mortality was collected using electronic medical records. Plasma C5a, TCC, FH, and ApoE were considered as a complement milieu. Tissue necrosis (HMGB1, RAGE), non-specific inflammatory responses (IL-6, C-reactive protein), overall viral burden (SARS-CoV-2 spike protein), and specific immune responses (IgG, IgA, IgM directed αS- & N-proteins) were assessed simultaneously. C5a remained elevated across all time points, with the peak at 5-7 days. Studied elements of complement coalesced around three clusters: #0 (↑↑↑C5a, ↑↑TCC, ↓↓ApoE), #1 ↑C5a, ↑TCC, ↑↑↑FH); #2 (↑C5a, ↑TCC, ↑FH, ↑↑↑ApoE). The decline in FH and ApoE was a predictor of death, while TCC and C5a correlated with patient length of stay, APACHE, and CRP. Increased levels of C5a (Δ = 122.64; p = 0.0294; data not shown) and diminished levels of FH (Δ = 836,969; p = 0.0285; data not shown) co-existed with CVA incidence. C5a correlated storngly with blood RAGE and HMGB1, but not with viral load and immunological responsiveness. Remdesivir positively affected FH preservation, while convalescent plasma treatment elevated C5a levels. Three clusters of complement activation demonstrated a various milieu of ApoE & FH vs C5a & TCC in COVID-19 patients. Complement activation is linked to increased necrosis markers but not to viral burden or immune system response.

A heterogeneous multi-modal medical data fusion framework supporting hybrid data exploration.

Industry 4.0 era has witnessed that more and more high-tech and precise devices are applied into medical field to provide better services. Besides EMRs, medical data include a large amount of unstructured data such as X-rays, MRI scans, CT scans and PET scans, which is still continually increasing. These massive, heterogeneous multi-modal data bring the big challenge to finding valuable data sets for healthcare researchers and other users. The traditional data warehouses are able to integrate the data and support interactive data exploration through ETL process. However, they have high cost and are not real-time. Furthermore, they lack of the ability to deal with multi-modal data in two phases-data fusion and data exploration. In the data fusion phase, it is difficult to unify the multi-modal data under one data model. In the data exploration phase, it is challenging to explore the multi-modal data at the same time, which impedes the process of extracting the diverse information underlying multi-modal data. Therefore, in order to solve these problems, we propose a highly efficient data fusion framework supporting data exploration for heterogeneous multi-modal medical data based on data lake. This framework provides a novel and efficient method to fuse the fragmented multi-modal medical data and store their metadata in the data lake. It offers a user-friendly interface supporting hybrid graph queries to explore multi-modal data. Indexes are created to accelerate the hybrid data exploration. One prototype has been implemented and tested in a hospital, which demonstrates the effectiveness of our framework.

A decline of protective apolipoprotein J and complement factor H concomitant with increase in C5a 3 months after cardiac surgery-Evidence of long-term complement perturbations.

Background: Heart surgery results in complement activation with the potential for collateral end-organ damage, especially if the protective elements (complement factor H, Apolipoprotein J) are inadequate. Here, we have investigated if peri-operative stress results in an imbalance between complement activation and its protective mechanisms up to 3 months after heart surgery. Methods: 101 patients scheduled for non-emergent cardiac surgery donated blood before the procedure (tbaseline), and 24 h (t24h ), 7 days (t7d ) and 3 months (t3m ) after. Complement activation was measured as a serum level of soluble activated component 5 (sC5a) and soluble terminal complement complex (sTCC). Simultaneously, protective complement factor H (CfH), and apolipoprotein J (ApoJ) were measured. Inflammatory responses were quantified using C-reactive protein (CRP) and interleukin-6 (IL-6). Details regarding anesthesia, intensive care unit (ICU) stay, pre-existing conditions, the incidence of postoperative complications, and mortality were collected from medical records. Results: C5a declined at t24h to rebound at t7d and t3m . sTCC was significantly depressed at t24h and returned to baseline at later time points. In contrast, CfH and ApoJ were depressed at t3m . Milieu of complement factors aligned along two longitudinal patterns:cluster#1 (C5a/sTTC continuously increasing and CfH/ApoJ preserved at tbaseline) and cluster#2 (transient sC5a/sTTC increase and progressive decline of CfH). Most patients belonged to cluster #1 at t24h (68%), t7d (74%) and t3m (72%). sTCC correlated with APACHE1h (r 2 =-0.25; p < 0.031) and APACHE24h (r 2 = 0.27; p < 0.049). IL-6 correlated with C5a (r 2 =-0.28; p < 0.042) and sTTC (r 2 =-0.28; p < 0.015). Peri-operative administration of acetaminophen and aspirin altered the complement elements. Prolonged hospital stay correlated with elevated C5a [t (78) = 2.03; p = 0.048] and sTTC serum levels [U (73) = 2.07; p = 0.037]. Patients with stroke had a decreased serum level of C5a at t7d and t3m. Conclusion: There is a significant decrease in complement protective factors 3 months after cardiac surgery, while C5a seems to be slightly elevated, suggesting that cardiac surgery affects complement milieu long into recovery.

Efficacy of an Experimental Gonococcal Lipooligosaccharide Mimitope Vaccine Requires Terminal Complement.

A safe and effective vaccine against multidrug-resistant gonorrhea is urgently needed. An experimental peptide vaccine called TMCP2 that mimics an oligosaccharide epitope in gonococcal lipooligosaccharide, when adjuvanted with glucopyranosyl lipid adjuvant-stable emulsion, elicits bactericidal immunoglobulin G and hastens clearance of gonococci in the mouse vaginal colonization model. In this study, we show that efficacy of TMCP2 requires an intact terminal complement pathway, evidenced by loss of activity in C9-/- mice or when C7 function was blocked. In conclusion, TMCP2 vaccine efficacy in the mouse vagina requires membrane attack complex. Serum bactericidal activity may serve as a correlate of protection for TMCP2.

Depleted histone deacetylase 3 or restored microRNA-19b-1-5p facilitates recovery of spinal cord injury via inactivating JAK2/STAT3 signaling pathway.

We intended to discuss the influence of histone deacetylase 3 (HDAC3) on spinal cord injury (SCI) by regulating microRNA-19b-1-5p (miR-19b-1-5p) and janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) pathway. In a rat model, the role of HDAC3 and miR-19b-1-5p in SCI was identified through detecting motor function, serum inflammation, pathological damage, cell apoptosis and GFAP expression. Also, by measuring GFAP expression and migration of spinal cord astrocytes, the effects of HDAC3 and miR-19b-1-5p in SCI were identified in vitro. Restoration of miR-19b-1-5p or depletion of HDAC3 alleviated motor function, inflammation, relieved pathological damage and reduced apoptosis, and reduced GFAP expression in the spinal cord tissue of SCI rats. Up-regulating miR-19b-1-5p or down-regulating HDAC3 decreased migration and GFAP expression of injured astrocytes. Our study presents that down-regulated HDAC3 can facilitate the recovery of SCI via inhibiting the activation of JAK2/STAT3 pathway by up-regulating miR-19b-1-5p.

Classification of electrocardiogram signals with waveform morphological analysis and support vector machines.

Electrocardiogram (ECG) indicates the occurrence of various cardiac diseases, and the accurate classification of ECG signals is important for the automatic diagnosis of arrhythmia. This paper presents a novel classification method based on multiple features by combining waveform morphology and frequency domain statistical analysis, which offer improved classification accuracy and minimise the time spent for classifying signals. A wavelet packet is used to decompose a denoised ECG signal, and the singular value, maximum value, and standard deviation of the decomposed wavelet packet coefficients are calculated to obtain the frequency domain feature space. The slope threshold method is applied to detect R peak and calculate RR intervals, and the first two RR intervals are extracted as time-domain features. The fusion feature space is composed of time and frequency domain features. A combination of support vector machine (SVM) with the help of grid search and waveform morphological analysis is applied to complete nine types of ECG signal classification. Computer simulations show that the accuracy of the proposed algorithm on multiple types of arrhythmia databases can reach 96.67%.

Combating Complement's Deleterious Effects on Nanomedicine by Conjugating Complement Regulatory Proteins to Nanoparticles.

Complement opsonization is among the biggest challenges facing nanomedicine. Nearly instantly after injection into blood, nanoparticles are opsonized by the complement protein C3, leading to clearance by phagocytes, fouling of targeting moieties, and release of anaphylatoxins. While surface polymers such as poly(ethylene glycol) (PEG) partially decrease complement opsonization, most nanoparticles still suffer from extensive complement opsonization, especially when linked to targeting moieties. To ameliorate the deleterious effects of complement, two of mammals' natural regulators of complement activation (RCAs), Factors H and I, are here conjugated to the surface of nanoparticles. In vitro, Factor H or I conjugation to PEG-coated nanoparticles decrease their C3 opsonization, and markedly reduce nanoparticle uptake by phagocytes. In an in vivo mouse model of sepsis-induced lung injury, Factor I conjugation abrogates nanoparticle uptake by intravascular phagocytes in the lungs, allowing the blood concentration of the nanoparticle to remain elevated much longer. For nanoparticles targeted to the lung's endothelium by conjugation to anti-ICAM antibodies, Factor I conjugation shifts the cell-type distribution away from phagocytes and toward endothelial cells. Finally, Factor I conjugation abrogates the severe anaphylactoid responses common to many nanoparticles, preventing systemic capillary leak and preserving blood flow to visceral organs and the brain. Thus, conjugation of RCAs, like Factor I, to nanoparticles is likely to help in nanomedicine's long battle against complement, improving several key parameters critical for clinical success.

Crosstalk between Heart Failure and Cognitive Impairment via hsa-miR-933/RELB/CCL21 Pathway.

BACKGROUND: The association between heart failure (HF) and cognitive impairment has received increasing attention from scholars and researchers in recent years. However, no systematic studies have been carried out yet focused on the crosstalk between heart failure and cognitive impairment via miRNAs. METHODS: GSE104150, GSE53473, GSE120584, and GSE116250 with RNA-seq data and clinical data were downloaded from the GSE database. All data were statistically analysed using R software to detect DE-miRNAs and DE-mRNAs associated with both HF and cognitive impairment. Protein-protein interaction (PPI) networks were mapped, and a logistic regression model for cognitive impairment prediction was developed. Furthermore, the TTRUST database and miRWalk were used to map miRNA-transcription factor (TF) and messenger RNA (mRNA) regulatory pathways. Finally, core TFs were enriched for analysis. RESULTS: Differentially enriched DE-miRNAs and DE-mRNAs both present in HF and cognitive impairment were determined. A logistic regression model established based on DE-miRNAs was validated to have a strong performance in cognitive impairment prediction. The core miRNA-TF-mRNA pathway was formed by mapping the PPI networks associated with the two diseases. Further GSEA was performed with V-rel reticuloendotheliosis viral oncogene homolog B (RELB) as the core TF, and the retinol metabolism and gap junction pathways were analysed. CONCLUSIONS: This study was the first attempt to predict the crosstalk and examine underlying mechanisms between HF and cognitive impairment applying bioinformatics. The findings suggested a potential hsa-miR-933/RELB/CCL21 regulatory axis correlated with HF and neurological disorders (or cognitive impairment), according to PPI networks.

Artificial Intelligence-Enabled ECG Algorithm Based on Improved Residual Network for Wearable ECG.

Heart disease is the leading cause of death for men and women globally. The residual network (ResNet) evolution of electrocardiogram (ECG) technology has contributed to our understanding of cardiac physiology. We propose an artificial intelligence-enabled ECG algorithm based on an improved ResNet for a wearable ECG. The system hardware consists of a wearable ECG with conductive fabric electrodes, a wireless ECG acquisition module, a mobile terminal App, and a cloud diagnostic platform. The algorithm adopted in this study is based on an improved ResNet for the rapid classification of different types of arrhythmia. First, we visualize ECG data and convert one-dimensional ECG signals into two-dimensional images using Gramian angular fields. Then, we improve the ResNet-50 network model, add multistage shortcut branches to the network, and optimize the residual block. The ReLu activation function is replaced by a scaled exponential linear units (SELUs) activation function to improve the expression ability of the model. Finally, the images are input into the improved ResNet network for classification. The average recognition rate of this classification algorithm against seven types of arrhythmia signals (atrial fibrillation, atrial premature beat, ventricular premature beat, normal beat, ventricular tachycardia, atrial tachycardia, and sinus bradycardia) is 98.3%.

Erythrocytes identify complement activation in patients with COVID-19.

COVID-19, the disease caused by the SARS-CoV-2 virus, can progress to multisystem organ failure and viral sepsis characterized by respiratory failure, arrhythmias, thromboembolic complications, and shock with high mortality. Autopsy and preclinical evidence implicate aberrant complement activation in endothelial injury and organ failure. Erythrocytes express complement receptors and are capable of binding immune complexes; therefore, we investigated complement activation in patients with COVID-19 using erythrocytes as a tool to diagnose complement activation. We discovered enhanced C3b and C4d deposition on erythrocytes in COVID-19 sepsis patients and non-COVID sepsis patients compared with healthy controls, supporting the role of complement in sepsis-associated organ injury. Our data suggest that erythrocytes may contribute to a precision medicine approach to sepsis and have diagnostic value in monitoring complement dysregulation in COVID-19-sepsis and non-COVID sepsis and identifying patients who may benefit from complement targeted therapies.

von Willebrand factor variants in C3 glomerulopathy: A Chinese cohort study.

C3 glomerulopathy (C3G) is a rare renal disease characterized by predominant glomerular C3 staining. Complement alternative pathway dysregulation due to inherited complement defects is associated with C3G. To identify novel C3G-related genes, we screened 86 genes in the complement, coagulation and endothelial systems in 35 C3G patients by targeted genomic enrichment and massively parallel sequencing. Surprisingly, the most frequently mutated gene was VWF. Patients with VWF variants had significantly higher proteinuria levels, higher crescent formation and lower factor H (FH) levels. We further selected two VWF variants to transiently express the von Willebrand factor (vWF) protein, we found that vWF expression from the c.1519A > G variant was significantly reduced. In vitro results further indicated that vWF could regulate complement activation, as it could bind to FH and C3b, act as a cofactor for factor I-mediated cleavage of C3b. Thus, we speculated that vWF might be involved in the pathogenesis of C3G.

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The statistical model (log-normal model), niche models (Zipf model, broken stick mo-del, niche preemption model), and neutral model were used to fit the species-abundance distribution patterns based on the measurements of environmental factors and inventory data of trees with DBH≥1 cm in a 1.5 hm2 plot in the primary forest (PF) and a 1.5 hm2 plot in the secondary forest (SF). The results showed that species-abundance distribution was affected by habitat heterogeneity in Q. aliena var. acutiserrata forest. Topography had a predominant impact on the species-abundance distribution in PF. Species distribution was affected by both neutral and niche processes, with neutral process having a less prominent effect in large convexity habitats. While the neutral model was rejected by the K-S and Chi-square test in low convexity habitats, the species-abundance distribution satisfied the assumption of niche theory. Niche process and neutral process were equally important in the community in areas with steep slopes, while niche differentiation was the dominant in flat areas. In SF, the main factors affecting species distribution were soil nutrients. The niche process was the mainly ecological process affected species-abundance distribution in habitats with high soil available phosphorus, while the niche and neutral processes existed simultaneously in habitats with low soil phosphorus availability. There was a significant scale effect on the species-abundance distribution pattern of Q. aliena var. acutiserrata forests in Taibai Mountain. The niche and neutral processes could protect the species-abundance distribution at the 20 m×20 m scale in PF, while the niche process could explain the species-abundance distribution at the 40 m×40 m and 70 m×70 m scales. The niche and neutral processes combined acted on the species abundance distribution at the 20 m×20 m, 40 m×40 m and 70 m×70 m scales in SF, with niche process being more important than neutral process. Moreover, besides the scale and habitat heterogeneity, the species-abundance distribution patterns of Q. aliena var. acutiserrata forests differed significantly between primary forest and secondary forest under anthropogenic disturbance.

Preparation, characterization, and catalytic activity of a novel MgO/expanded graphite for ozonation of Cu-EDTA.

Magnesium oxide/expanded graphite (MgO/EG) catalyst was synthesized and applied for enhancing the degradation of Cu-ethylenediaminetetraacetic acid (Cu-EDTA) in an aqueous solution. The MgO/EG catalyst was characterized by XRD, SEM, EDS, and FTIR. For assessing the catalytic activity of MgO/EG, essential influencing factors were investigated including catalyst dosage, O3 dosage, initial pH, initial Cu-EDTA concentration, and coexisting ions. The results show that the catalytic material showed high catalytic oxidation capacity for the Cu-EDTA removal in the MgO/EG/O3 system. 100% of Cu(II) and 73.2% of TOC removal efficiency could be achieved in the MgO/EG/O3 system at the reaction times of 90 min. This efficiency was higher than that seen for other systems, including O3 alone (Cu(II) 81.4%/TOC 60.6%), EG/O3 (84.2%/64.1), MgO/EG (< 4%/< 4%), and EG (< 4%/< 4%). A small decrease in the Cu(II) and TOC removal rate was observed after three runs in the stability and reusability experiments of the catalyst. Assays with radical scavenging experiments confirmed that MgO/EG-mediated oxidation was dependent on a hydroxyl radical pathway. The UV-vis spectra confirmed that the absorption peak of Cu-EDTA was gradually decreased and finally disappeared.

Development of Complement Factor H-Based Immunotherapeutic Molecules in Tobacco Plants Against Multidrug-Resistant Neisseria gonorrhoeae.

Novel therapeutics against the global threat of multidrug-resistant Neisseria gonorrhoeae are urgently needed. Gonococci possess several mechanisms to evade killing by human complement, including binding of factor H (FH), a key inhibitor of the alternative pathway. FH comprises 20 short consensus repeat (SCR) domains organized in a head-to-tail manner as a single chain. N. gonorrhoeae binds two regions in FH; domains 6 and 7 and domains 18 through 20. We designed a novel anti-infective immunotherapeutic molecule that fuses domains 18-20 of FH containing a D-to-G mutation in domain 19 at position 1119 (called FH*) with human IgG1 Fc. FH*/Fc retained binding to gonococci but did not lyse human erythrocytes. Expression of FH*/Fc in tobacco plants was undertaken as an alternative, economical production platform. FH*/Fc was expressed in high yields in tobacco plants (300-600 mg/kg biomass). The activities of plant- and CHO-cell produced FH*/Fc against gonococci were similar in vitro and in the mouse vaginal colonization model of gonorrhea. The addition of flexible linkers [e.g., (GGGGS)2 or (GGGGS)3] between FH* and Fc improved the bactericidal efficacy of FH*/Fc 2.7-fold. The linkers also improved PMN-mediated opsonophagocytosis about 11-fold. FH*/Fc with linker also effectively reduced the duration and burden of colonization of two gonococcal strains tested in mice. FH*/Fc lost efficacy: i) in C6-/- mice (no terminal complement) and ii) when Fc was mutated to abrogate complement activation, suggesting that an intact complement was necessary for FH*/Fc function in vivo. In summary, plant-produced FH*/Fc represent promising prophylactic or adjunctive immunotherapeutics against multidrug-resistant gonococci.

Erythrocytes Reveal Complement Activation in Patients with COVID-19.

COVID-19, the disease caused by the SARS-CoV-2 virus, can progress to multi-organ failure characterized by respiratory insufficiency, arrhythmias, thromboembolic complications and shock. The mortality of patients hospitalized with COVID-19 is unacceptably high and new strategies are urgently needed to rapidly identify and treat patients at risk for organ failure. Clinical epidemiologic studies demonstrate that vulnerability to organ failure is greatest after viral clearance from the upper airway, which suggests that dysregulation of the host immune response is a critical mediator of clinical deterioration and death. Autopsy and pre-clinical evidence implicate aberrant complement activation in endothelial injury and organ failure. A potential therapeutic strategy warranting investigation is to inhibit complement, with case reports of successful treatment of COVID-19 with inhibitors of complement. However, this approach requires careful balance between the host protective and potential injurious effects of complement activation, and biomarkers to identify the optimal timing and candidates for therapy are lacking. Here we report the presence of complement activation products on circulating erythrocytes from hospitalized COVID-19 patients using flow cytometry. These findings suggest that novel erythrocyte-based diagnostics provide a method to identify patients with dysregulated complement activation.