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

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

C5b-9 promotes ferritinophagy leading to ferroptosis in renal tubular epithelial cells of trichloroethylene-sensitized mice.

Trichloroethylene (TCE) is a common environmental contaminant that can cause a severe allergic reaction called TCE hypersensitivity syndrome, which often implicates the patient's kidneys. Our previous study revealed that C5b-9-induced tubular ferroptosis is involved in TCE-caused kidney damage. However, the study did not explain how tubule-specific C5b-9 causes free iron overload, a key event in ferroptosis. Here, we aimed to explore the role of NCOA4-mediated ferritinophagy in C5b-9-induced iron overload and ferroptosis in TCE-sensitized mice. Our results showed that TCE sensitization does not affect iron import or export, but does affect iron storage, causing ferritin degradation and free iron overload. In addition, mitochondrial ROS was upregulated, and these changes were blocked by C5b-9 inhibition. Interestingly, TCE-induced ferritin degradation and ferroptosis were significantly antagonized by the application of the mitochondrial ROS inhibitor, Mito-TEMPO. Moreover, all of these modes of action were further verified in C5b-9-attack signalling HK-2 cells. Further investigation demonstrated that C5b-9-upregulated mitochondrial ROS induced a marked increase in nuclear receptor coactivator 4 (NCOA4), a master regulator of ferritinophagy. In addition, the application of NCOA4 small interfering RNA not only significantly reversed ferritinophagy caused by C5b-9 but also reduced C5b-9-induced ferroptosis in HK-2 cells. Taken together, these results suggest that tubule-specific C5b-9 deposition activates NCOA4 through the upregulation of mitochondrial ROS, causing ferritin degradation and elevated free iron, which ultimately leads to tubular epithelial cell ferroptosis and kidney injury in TCE-sensitized mice.

Pharmacodynamics and Mechanism of Astragali Radix and Anemarrhenae Rhizoma in Treating Chronic Heart Failure by Inhibiting Complement Activation.

Astragali radix (AR) and anemarrhenae rhizoma (AAR) are used clinically in Chinese medicine for the treatment of chronic heart failure (CHF), but the exact therapeutic mechanism is unclear. In this study, a total of 60 male C57BL/6 mice were divided into 5 groups, namely sham, model, AR, AAR, and AR-AAR. In the sham group, the chest was opened without ligation. In the other groups, the chest was opened and the transverse aorta was ligated to construct the transverse aortic constriction model. After 8 weeks of feeding, mice were given medicines by gavage for 4 weeks. Left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) were detected by echocardiography. Heart weight index (HWI) and wheat germ agglutinin staining were used to evaluate cardiac hypertrophy. Hematoxylin-eosin staining was used to observe the pathological morphology of myocardial tissue. Masson staining was used to evaluate myocardial fibrosis. The content of serum brain natriuretic peptide (BNP) was detected by enzyme-linked immunosorbent assay kit. The content of serum immunoglobulin G (IgG) was detected by immunoturbidimetry. The mechanism of AR-AAR in the treatment of CHF was explored by proteomics. Western blot was used to detect the protein expressions of complement component 1s (C1s), complement component 9 (C9), and terminal complement complex 5b-9 (C5b-9). The results show that AR-AAR inhibits the expression of complement proteins C1s, C9, and C5b-9 by inhibiting the production of IgG antibodies from B cell activation, which further inhibits the complement activation, attenuates myocardial fibrosis, reduces HWI and cardiomyocyte cross-sectional area, improves cardiomyocyte injury, reduces serum BNP release, elevates LVEF and LVFS, improves cardiac function, and exerts myocardial protection.

Forensic significance of intracardiac expressions of Nrf2 in acute myocardial ischemia.

When exposed to oxidative and electrophilic stress, a protective antioxidant response is initiated by nuclear factor erythroid 2-related factor 2 (Nrf2). However, the extent of its importance in the forensic diagnosis of acute ischemic heart diseases (AIHD), such as myocardial infarction (MI), remains uncertain. On the other hand, immunohistochemical analyses of fibronectin (FN) and the terminal complement complex (C5b-9) prove valuable in identifying myocardial ischemia that precedes necrosis during the postmortem diagnosis of sudden cardiac death (SCD). In this study, we investigated the immunohistochemical levels of Nrf2, FN, and C5b-9 in human cardiac samples to explore their forensic relevance for the identification of acute cardiac ischemia. Heart samples were obtained from 25 AIHD cases and 39 non-AIHD cases as controls. Nrf2 was localized in the nuclei of cardiomyocytes, while FN and C5b-9 were detected in the myocardial cytoplasm. The number of intranuclear Nrf2 positive signals in cardiomyocytes increased in AIHD cases compared to control cases. Additionally, the grading of positive portions of cardiac FN and C5b-9 in the myocardium was also significantly enhanced in AIHD, compared to controls. Collectively, these results indicate that the immunohistochemical investigation of Nrf2 combined with FN, and/or C5b-9 holds the potential for identifying early-stage myocardial ischemic lesions in cases of SCD.

An up-to-date myopathologic characterisation of facioscapulohumeral muscular dystrophy type 1 muscle biopsies shows sarcolemmal complement membrane attack complex deposits and increased skeletal muscle regeneration.

The aim of this study was to identify key routinely used myopathologic biomarkers of FSHD1. Needle muscle biopsies were taken in 34 affected muscles (m. quadriceps femoris (QF), n = 20, m. tibialis anterior (TA), n = 13, m. biceps brachii, n = 1) from 22 patients (age, 53.5 (10) years; M = 12, F = 10). Eleven patients had more than one biopsy (2xQF, n = 1; QF+TA, n = 9; 2xQF+TA, n = 1). Histochemistry, immunoperoxidase, and immunofluorescence stainings were performed and compared to age and muscle type matched muscle specimens of 11 healthy controls. Myopathologic features observed in our FSHD1 cohort were internalized nuclei, type 1 fibre hypertrophy and NADH central clearances/cores. We observed a prominent inflammatory response with MAC deposits, MHC I expression, and muscle regeneration that correlated with the inflammatory score. Our up-to-date characterization of FSHD1 points towards MHC I, MAC, and embryonic Myosin Heavy Chain/muscle regeneration as useful myopathologic readouts of FSHD1.

Complement C7 and clusterin form a complex in circulation.

Introduction: The complement system is part of innate immunity and is comprised of an intricate network of proteins that are vital for host defense and host homeostasis. A distinct mechanism by which complement defends against invading pathogens is through the membrane attack complex (MAC), a lytic structure that forms on target surfaces. The MAC is made up of several complement components, and one indispensable component of the MAC is C7. The role of C7 in MAC assembly is well documented, however, inherent characteristics of C7 are yet to be investigated. Methods: To shed light on the molecular characteristics of C7, we examined the properties of serum-purified C7 acquired using polyclonal and novel monoclonal antibodies. The properties of serum­purified C7 were investigated through a series of proteolytic analyses, encompassing Western blot and mass spectrometry. The nature of C7 protein-protein interactions were further examined by a novel enzyme-linked immunosorbent assay (ELISA), as well as size­exclusion chromatography. Results: Protein analyses showcased an association between C7 and clusterin, an inhibitory complement regulator. The distinct association between C7 and clusterin was also demonstrated in serum-purified clusterin. Further assessment revealed that a complex between C7 and clusterin (C7-CLU) was detected. The C7-CLU complex was also identified in healthy serum and plasma donors, highlighting the presence of the complex in circulation. Discussion: Clusterin is known to dissociate the MAC structure by binding to polymerized C9, nevertheless, here we show clusterin binding to the native form of a terminal complement protein in vivo. The presented data reveal that C7 exhibits characteristics beyond that of MAC assembly, instigating further investigation of the effector role that the C7-CLU complex plays in the complement cascade.

A novel flavonol-polysaccharide from Tamarix chinensis alleviates influenza A virus-induced acute lung injury. Evidences for its mechanism of action.

BACKGROUND: Tamarix chinensis Lour. is a Chinese medicine used for treating inflammation-related diseases and its crude polysaccharides (MBAP90) exhibited significant anticomplement activities in vitro. PURPOSE: To obtain anticomplement homogenous polysaccharides from MBAP90 and explore its therapeutic effects and potential mechanism on influenza A virus (IAV)-induced acute lung injury (ALI). METHODS: Anticomplement activity-guided fractionation of the water-soluble crude polysaccharides from the leaves and twigs of T. chinensis were performed by diethylaminoethyl-52 (DEAE-52) cellulose and gel permeation columns to yield a homogeneous polysaccharide MBAP-5, which was further characterized using ultra-high-performance liquid chromatography-ion trap tandem mass spectrometry (UPLC-IT-MS) and nuclear magnetic resonance (NMR) analysis. In vitro, the anticomplement activity of MBAP-5 through classical pathway was measured using a hemolytic test. The therapeutic effects of MBAP-5 on ALI were evaluated in H1N1-infected mice. H&E staining, enzyme linked immunosorbent assay (ELISA), immunohistochemistry, and western blot were used to systematically access lung histomorphology, inflammatory cytokines, degree of complement component 3c, 5aR, and 5b-9 (C3c, C5aR, and C5b-9) deposition, and inflammasome signaling pathway protein expressions in lung tissues. RESULTS: MBAP-5 was a novel flavonol-polysaccharide with the molecular weight (Mw) of 153.6 kDa. Its structure was characterized to process a backbone of →4)-α-D-GlcpA-(1→, →6)-α-D-Glcp-(1→, →3,4)-α-D-Glcp-(1→, →3,4,6)-α-D-Glcp-(1→, and →4,6)-ß-D-Glcp-(1→, as well as branches of α-L-Araf-(1→ and ß-D-Galp-(1→. Particularly, O-3 of →3,4,6)-α-D-Glcp-(1→ was substituted by quercetin. In vitro assay showed that MBAP-5 had a potent anticomplement activity with a CH50 value of 102 ± 4 µg/ml. Oral administration of MBAP-5 (50 and 100 mg/kg) effectively attenuated the H1N1-induced pulmonary injury in vivo by reducing pulmonary edema, virus replication, and inflammatory responses. Mechanistically, MBAP-5 inhibited the striking deposition and contents of complement activation products (C3c, C5aR, and C5b-9) in the lung. Toll-like receptor 4 (TLR4) /transcription factor nuclear factor κB (NF-κB) signaling pathway was constrained by MBAP-5 treatment. In addition, MBAP-5 could suppress activation of the inflammasome pathways, including Nod-like receptor pyrin domain 3 (NLRP3), cysteinyl aspartate specific proteinase-1/12 (caspase-1/12), apoptosis­associated speck­like protein (ASC), gasdermin D (GSDMD), interleukin (IL)-1ß, and IL-18 expressions. CONCLUSIONS: A novel flavonol-polysaccharide MBAP-5 isolated from T. chinensis demonstrated a therapeutic effect against ALI induced by IAV attack. The mechanism might be associated with inhibition of complement system and inflammasome pathways activation.

The activated complement pathway in the fibrous process of benign prostatic hyperplasia.

BACKGROUND: To elucidate the changes in activated complement pathway in the fibrous process of benign prostatic hyperplasia (BPH), we analyzed the correlation between complement component expression and histological types of fibrosis using human BPH tissue. METHODS: Fifty-six histological BPH patients who underwent prostate needle biopsy at our institution (mean age 68.6 ± 6.5 years), divided into two histological groups, fibromuscular and fibrous, were compared. Inflammatory cell infiltration in BPH tissue was evaluated by immunohistochemical staining using CD45, with complement expression analysis performed using C3, factor B, and C5b-9 antibody, and the occupancy ratio of the stained region was calculated. Further, correlation between the histological types of fibrous components in BPH tissue and lower urinary tract symptoms questionnaires was analyzed. RESULTS: Twenty-seven (48.2%) and 29 (51.8%) cases were classified in the fibromuscular and fibrous groups, respectively. The proportion of CD45-positive cells in BPH tissue was significantly higher in the fibromuscular group. In complement component analysis, factor B did not significantly differ between groups, while C3 (fibromuscular group; 10.7 ± 8.2%, fibrous group; 16.4 ± 12.7%) and C5b-9 (fibromuscular group; 15.9 ± 6.2%, fibrous group; 17.6 ± 9.2%) were significantly higher in the fibrous group (p = 0.04, p = 0.04, respectively). International Prostate Symptom Score Q5 subscore, indicating slow stream, was significantly higher in the fibrous group (p = 0.04). CONCLUSIONS: In fibrous BPH with abundant fibrosis, the late complement pathway in addition to alternative pathway was activated compared to fibromuscular BPH. These results suggested that the alternative and late complement pathways were involved in the histological fibrous process of BPH.

ERK1/2-dependent activity of SOX9 is required for sublytic C5b-9-induced expression of FGF1, PDGFα, and TGF-ß1 in rat Thy-1 nephritis.

Mesangial proliferative glomerulonephritis (MsPGN) and its related rat model Thy-1 nephritis (Thy-1N) are associated with C5b-9 deposition and are characterized by proliferation of glomerular mesangial cell (GMC) and expansion of extracellular matrix (ECM) expansion, alongside overexpression of multiple growth factors. Although fibroblast growth factor 1 (FGF1), platelet-derived growth factor alpha (PDGFα), and transforming growth factor beta 1 (TGF-ß1) are well known for their proproliferative and profibrotic roles, the molecular mechanisms responsible for regulating the expression of these growth factors have not been thoroughly elucidated. In this study, we found that sublytic C5b-9 induction of sex-determining region Y-box 9 (SOX9) transactivated FGF1, PDGFα, and TGF-ß1 genes in GMCs, resulting in a significant increase in their mRNA and protein levels. Besides, sublytic C5b-9 induction of activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) phosphorylated SOX9 at serine 181 and serine 64, which enhanced SOX9's ability to transactivate FGF1, PDGFα, and TGF-ß1 genes in GMCs. Furthermore, we demonstrated that inhibiting ERK1/2 activation or silencing either ERK1/2 or SOX9 gene led to reduced SOX9 phosphorylation, decreased generation of FGF1, PDGFα, and TGF-ß1, and ameliorated glomerular injury in rat Thy-1N. Overall, these findings suggest that expression of FGF1, PDGFα, and TGF-ß1 is promoted by ERK1/2-mediated phosphorylation of SOX9, which may provide a valuable insight into the pathogenesis of MsPGN and offer a potential target for the development of novel treatment strategies for MsPGN.

Differential contributions of the C5b-9 and C5a/C5aR pathways to microvascular and macrovascular thrombosis in complement-mediated thrombotic microangiopathy patients.

To clarify the role of the C5a/C5aR (C5a receptor) and C5b-9 pathways in macrovascular thrombosis (MAT) and renal microthrombosis (MIT), 73 renal biopsy-proven complement-mediated thrombotic microangiopathy (C-TMA) patients were enrolled; 9 patients with pure MAT and 13 patients with pure MIT were selected for further study. Twenty-five external C-TMA patients were selected as the validation cohort. Plasma C5a and sC5b-9 (soluble C5b-9) levels were significantly higher in patients with MAT than in those with MIT (P = 0.008, P = 0.041, respectively). The mean optical density of C5aR1 in the kidney was significantly higher in MAT patients than in those with MIT (P < 0.001). Both urinary sC5b-9 levels (MIT: P < 0.001, MAT: P = 0.004) and renal deposition of C5b-9 (MIT: P < 0.001, MAT: P = 0.001) were significantly higher in C-TMA patients compared to normal control, but were similar between MAT and MIT groups. In the correlation analysis within 22C-TMA patients, urinary sC5b-9 levels and renal deposition of C5b-9 were positively correlated to renal MIT formation (P = 0.009 and P = 0.031, respectively). Furthermore, the renal citrullinated histone H3 (CitH3)- and neutrophil elastase (NE)-positive area ratios were both significantly higher in the MAT group than in the MIT group (P = 0.006 and P = 0.020, respectively). Therefore, the local C5b-9 and C5a/C5aR1 pathways might have differential contributions to MIT and MAT formation in the disease.

Voluntary wheel running prevents formation of membrane attack complexes and myelin degradation after peripheral nerve injury.

Regular aerobic activity is associated with a reduced risk of chronic pain in humans and rodents. Our previous studies in rodents have shown that prior voluntary wheel running can normalize redox signaling at the site of peripheral nerve injury, attenuating subsequent neuropathic pain. However, the full extent of neuroprotection offered by voluntary wheel running after peripheral nerve injury is unknown. Here, we show that six weeks of voluntary wheel running prior to chronic constriction injury (CCI) reduced the terminal complement membrane attack complex (MAC) at the sciatic nerve injury site. This was associated with increased expression of the MAC inhibitor CD59. The levels of upstream complement components (C3) and their inhibitors (CD55, CR1 and CFH) were altered by CCI, but not increased by voluntary wheel running. Since MAC can degrade myelin, which in turn contributes to neuropathic pain, we evaluated myelin integrity at the sciatic nerve injury site. We found that the loss of myelinated fibers and decreased myelin protein which occurs in sedentary rats following CCI was not observed in rats with prior running. Substitution of prior voluntary wheel running with exogenous CD59 also attenuated mechanical allodynia and reduced MAC deposition at the nerve injury site, pointing to CD59 as a critical effector of the neuroprotective and antinociceptive actions of prior voluntary wheel running. This study links attenuation of neuropathic pain by prior voluntary wheel running with inhibition of MAC and preservation of myelin integrity at the sciatic nerve injury site.

Up-regulation of scleral C5b-9 and its regulation of the NLRP3 inflammasome in a form-deprivation myopia mouse model.

BACKGROUND: Myopia has become a major public health problem worldwide. Although the involvement of the complement system in myopia progression has been reported, the underlying mechanism has not been well established. In this study, we induced a form deprivation (FD) myopia mouse model to investigate the mechanisms. METHODS: Both C6-knockout (KO) and wild-type (WT) mice were divided into FD and normal control (NC) groups. The FD myopia was induced in the right eyes of 24-day-old mice using a translucent balloon for 4 weeks. The left eye remained untreated and served as self-control. NC group received no treatment. Refractive error and axial length were measured at baseline, 2 weeks, and 4 weeks later under normal visual, 4 weeks after FD. Scleral transcriptome sequencing analysis was performed in in FD mice. The scleral levels of C5b-9, NLRP3, Caspase-1, IL-1ß, MMP-2, and collagen I were evaluated using immunohistochemistry. RESULTS: RNA-seq analysis showed 1058 differentially expressed genes. The GO analysis showed these genes were mainly related to the extracellular matrix, and immune response. The KEGG enrichment analysis showed that complement cascades were upregulated. Under normal visual conditions, both genotypes of mice exhibited comparable refractive error and axial length. However, after four weeks of FD, C6-KO mice showed a significantly less myopic shift (-2.28 ± 0.28 D versus -5.40 ± 1.33 D, P = 0.003), and axial shift (0.043 ± 0.032 mm versus 0.083 ± 0.026 mm, P = 0.042) in comparison to WT mice. Furthermore, the levels of C5b-9, NLRP3, caspase-1, IL-1ß, and MMP-2 were found to be elevated in the deprived eyes of WT mice in comparison to their fellow eyes, whereas the extent of this increase was significantly lower in C6-KO mice. CONCLUSIONS: Complement cascades are activated in FD myopia model. Upregulation of C5b-9 might participate in scleral remodeling during myopia progression via regulation of NLRP3 inflammasome activation.

Artificial surface labelling of Escherichia coli with StrepTagII antigen to study how monoclonal antibodies drive complement-mediated killing.

Antibodies play a key role in the immune defence against Gram-negative bacteria. After binding to bacterial surface antigens, IgG and IgM can activate the complement system and trigger formation of lytic membrane attack complex (MAC) pores. Molecular studies to compare functional activity of antibodies on bacteria are hampered by the limited availability of well-defined antibodies against bacterial surface antigens. Therefore, we genetically engineered E. coli by expressing the StrepTagII antigen into outer membrane protein X (OmpX) and validated that these engineered bacteria were recognised by anti-StrepTagII antibodies. We then combined this antigen-antibody system with a purified complement assay to avoid interference of serum components and directly compare MAC-mediated bacterial killing via IgG1 and pentameric IgM. While both IgG1 and IgM could induce MAC-mediated killing, we show that IgM has an increased capacity to induce complement-mediated killing of E. coli compared to IgG1. While Fc mutations that enhance IgG clustering after target binding could not improve MAC formation, mutations that cause formation of pre-assembled IgG hexamers enhanced the complement activating capacity of IgG1. Altogether, we here present a system to study antibody-dependent complement activation on E. coli and show IgM's enhanced capacity over IgG to induce complement-mediated lysis of E. coli.

Modeling complement activation on human glomerular microvascular endothelial cells.

Introduction: Atypical hemolytic uremic syndrome (aHUS) is a rare kidney disease caused by dysregulation of the complement alternative pathway. The complement dysregulation specifically leads to damage to the glomerular endothelium. To further understand aHUS pathophysiology, we validated an ex vivo model for measuring complement deposition on both control and patient human glomerular microvascular endothelial cells (GMVECs). Methods: Endothelial cells were incubated with human test sera and stained with an anti-C5b-9 antibody to visualize and quantify complement depositions on the cells with immunofluorescence microscopy. Results: First, we showed that zymosan-activated sera resulted in increased endothelial C5b-9 depositions compared to normal human serum (NHS). The levels of C5b-9 depositions were similar between conditionally immortalized (ci)GMVECs and primary control GMVECs. The protocol with ciGMVECs was further validated and we additionally generated ciGMVECs from an aHUS patient. The increased C5b-9 deposition on control ciGMVECs by zymosan-activated serum could be dose-dependently inhibited by adding the C5 inhibitor eculizumab. Next, sera from five aHUS patients were tested on control ciGMVECs. Sera from acute disease phases of all patients showed increased endothelial C5b-9 deposition levels compared to NHS. The remission samples showed normalized C5b-9 depositions, whether remission was reached with or without complement blockage by eculizumab. We also monitored the glomerular endothelial complement deposition of an aHUS patient with a hybrid complement factor H (CFH)/CFH-related 1 gene during follow-up. This patient had already chronic kidney failure and an ongoing deterioration of kidney function despite absence of markers indicating an aHUS flare. Increased C5b-9 depositions on ciGMVECs were observed in all samples obtained throughout different diseases phases, except for the samples with eculizumab levels above target. We then tested the samples on the patient's own ciGMVECs. The C5b-9 deposition pattern was comparable and these aHUS patient ciGMVECs also responded similar to NHS as control ciGMVECs. Discussion: In conclusion, we demonstrate a robust and reliable model to adequately measure C5b-9-based complement deposition on human control and patient ciGMVECs. This model can be used to study the pathophysiological mechanisms of aHUS or other diseases associated with endothelial complement activation ex vivo.

Microvascular C5b-9 deposition in non-lesional skin in patients with SLE and its correlation with active lupus nephritis: a prospective observational study.

OBJECTIVE: Tissue damage in lupus nephritis (LN) is mediated by activation of the classical complement pathway. Complement-mediated upregulation of endothelial cell adhesion molecules is seen in dermal blood vessels of non-lesional skin of patients with active lupus. In diseases with systemic complement activation, extensive microvascular C5b-9 deposition is seen in non-lesional skin. In this study, we assess the presence of systemic complement pathway activation as determined by non-lesional skin microvascular C5b-9 deposition in patients with LN. METHODS: Eight patients with active LN and eight patients without active LN underwent non-lesional skin biopsies. Using a diaminobenzidine technique, specimens were evaluated for microvascular C5b-9 consistent with systemic complement pathway activation. RESULTS: Five of eight patients with active LN and one of eight patients without active LN demonstrated positive C5b-9 staining in non-lesional skin (p=0.04). Positive non-lesional C5b-9 staining has greater specificity, 87.5%, for active LN than pyuria, low complements, elevated double-stranded DNA (dsDNA) and proteinuria. Urine protein creatinine ratio was significantly higher in patients with positive non-lesional C5b-9 deposition (5.18 vs 1.20; p=0.04). C5b-9 deposition was not associated with a higher NIH Activity Index, interstitial fibrosis, dsDNA or lower complements. CONCLUSION: This is the first study to demonstrate evidence in non-lesional skin of microvascular C5b-9 indicative of systemic complement pathway activation in LN. C5b-9 deposition is statistically more common and demonstrated greater specificity than most historical biomarkers for active LN. The findings support a potential role for microvascular C5b-9 assessment in non-lesional skin as a biomarker for LN activity.

Urine Proteomics Link Complement Activation with Interstitial Fibrosis/Tubular Atrophy in Lupus Nephritis Patients.

BACKGROUND: Intrarenal complement activation has been implicated in the pathogenesis of tubulointerstitial fibrosis in lupus nephritis (LN) based on prior animal studies. The assembly of the membrane attack complex (MAC) by complement C5b to C9 on the cell membrane leads to cytotoxic pores and cell lysis, while CD59 inhibits MAC formation by preventing C9 from joining the complex. We hypothesize that complement activation and imbalance between complement activation and inhibition, as defined by increased production of individual complement components and uncontrolled MAC activation relative to CD59 inhibition, are associated with interstitial fibrosis and tubular atrophy (IFTA) in LN and correlate with the key mediators of kidney fibrosis- transforming growth factor receptors beta (TGFRß), platelet-derived growth factor beta (PDGFß) and platelet-derived growth factor receptor beta (PDGFRß). METHODS: We included urine samples from 46 adults and pediatric biopsy-proven lupus nephritis patients who underwent clinically indicated kidney biopsies between 2010 and 2019. We compared individual urinary complement components and the urinary C9-to-CD59 ratio between LN patients with moderate/severe IFTA and none/mild IFTA. IFTA was defined as none/mild (<25% of interstitium affected) versus moderate/severe (≥ 25% of interstitium affected). Proteomics analysis was performed using mass spectrometry (Orbitrap Fusion Lumos, Thermo Scientific) and processed by the Proteome Discoverer. Urinary complement proteins enriched in LN patients with moderate/severe IFTA were correlated with serum creatinine, TGFßR1, TGFßR2, PDGFß, and PDGFRß. RESULTS: Of the 46 LN patients included in the study, 41 (89.1%) were women, 20 (43.5%) self-identified as Hispanic or Latino, and 26 (56.5%) self-identified as Black or African American. Ten of the 46 (21.7%) LN patients had moderate/severe IFTA on kidney biopsy. LN patients with moderate/severe IFTA had an increased urinary C9-to-CD59 ratio [median 0.91 (0.83-1.05) vs 0.81 (0.76-0.91), p=0.01]. Urinary C3 and CFI levels in LN patients with moderate/severe IFTA were higher compared to those with none/mild IFTA [C3 median (IQR) 24.4(23.5-25.5) vs. 20.2 (18.5-22.2), p= 0.02], [CFI medium (IQR) 28.8 (21.8-30.6) vs. 20.4 (18.5-22.9), p=0.01]. Complement C9, CD59, C3 and CFI correlated with TGFßR1, PDGFß, and PDGFRß, while C9, CD59 and C3 correlated with TGFßR2. CONCLUSION: This study is one of the first to compare the urinary complement profile in LN patients with moderate/severe IFTA and none/mild IFTA in human tissues. This study identified C3, CFI, and C9-to-CD59 ratio as potential markers of tubulointerstitial fibrosis in LN.

Alzheimer's Disease Biomarkers and Complement Proteins Mediate the Impact of Sleep Fragmentation on Cognitive Impairment in Obstructive Sleep Apnea Patients Without Dementia.

BACKGROUND: Cognitive impairment is common in patients with obstructive sleep apnea (OSA). Previous studies indicated that intermittent hypoxia, sleep fragmentation, and depressive symptoms were associated with cognitive impairment in OSA patients. OBJECTIVE: The study aimed to investigate whether sleep characteristics and depressive symptoms affected cognitive abilities mediated by Alzheimer's disease (AD) biomarkers and complement proteins in OSA patients without dementia. METHODS: A total of 317 subjects without dementia who had undergone polysomnography, cognitive and neuropsychological evaluations, were recruited. Neuronal-derived exosomes (NDEs) levels for amyloid-ß (Aß), total tau (T-tau), and tau phosphorylated 62 at threonine 181 (P-T181-tau) and astrocyte-derived exosomes (ADEs) levels for complement proteins were measured. Mediation analysis were performed to explore the mediation effects of AD biomarkers (Aß42, T-tau, P-T181-tau) and complement proteins (C3b and C5b-9) on cognition. RESULTS: The findings revealed that the association between sleep fragmentation and cognition was mediated by Aß42 (the percentage varied from 18.25% to 30.6%), P-T181-tau (the percentage varied from 24.36% to 32.3%), and C5b-9 (the percentage varied from 30.88% to 60.7%). The influence of depressive symptoms on cognition was only mediated via C3b (the percentage varied from 24.1% to 36.6%). CONCLUSIONS: In OSA patients without dementia, Aß42 and P-T181-tau levels in NDEs, and C5b-9 levels in ADEs mediated the impact of sleep fragmentation on cognitive impairment, and C3b levels in ADEs mediated the impact of depressive symptoms on cognitive impairment.

Genome-Wide Analysis of the Membrane Attack Complex and Perforin Genes and Their Expression Pattern under Stress in the Solanaceae.

The Membrane Attack Complex and Perforin (MACPF) proteins play a crucial role in plant development and adaptation to environmental stresses. Heretofore, few MACPF genes have been functionally identified, leaving gaps in our understanding of MACPF genes in other plants, particularly in the Solanaceae family, which includes economically and culturally significant species, such as tomato, potato, and pepper. In this study, we have identified 26 MACPF genes in three Solanaceae species and in the water lily, which serves as the base group for angiosperms. Phylogenetic analysis indicates that angiosperm MACPF genes could be categorized into three distinct groups, with another moss and spikemoss lineage-specific group, which is further supported by the examination of gene structures and domain or motif organizations. Through inter-genome collinearity analysis, it is determined that there are 12 orthologous SolMACPF gene pairs. The expansion of SolMACPF genes is primarily attributed to dispersed duplications, with purifying selection identified as the principal driving force in their evolutionary process, as indicated by the ω values. Furthermore, the analysis of expression patterns revealed that Solanaceae genes are preferentially expressed in reproductive tissues and regulated by various environmental stimuli, particularly induced by submergence. Taken together, these findings offer valuable insights into and a fresh perspective on the evolution and function of SolMACPF genes, thereby establishing a foundation for further investigations into their phenotypic and functional characteristics.

Characteristics of Complement Protein Deposition in Proliferative Glomerulonephritis with Monoclonal Immunoglobulin Deposition.

BACKGROUND: Hypocomplementemia and complement co-deposition with monoclonal immunoglobulins in glomeruli are not rare in proliferative glomerulonephritis with monoclonal immunoglobulin deposits (PGNMID). Deposition of monoclonal immunoglobulins in glomeruli has been suggested to activate complement and cause kidney injury. However, the profiles of complement activation in PGNMID and their clinical and pathologic significance need to be clarified. METHODS: Forty-six patients with PGNMID were enrolled. Proteomic analysis of glomeruli using laser microdissection and mass spectrometry was performed for ten patients with PGNMID to determine the composition of glomerular deposits. Kidney deposition of complement components was detected by immunohistochemistry and immunofluorescence. Urinary and plasma levels of complement components were measured by an enzyme-linked immunosorbent assay. Group differences were assessed using t tests or Mann-Whitney U tests depending on the distribution. Correlation analysis was performed using Spearman rank correlation or Pearson correlation. RESULTS: Laser microdissection and mass spectrometry-based proteomic analysis showed that complement components were the most enriched proteins deposited in the glomeruli of patients with PGNMID. Glomerular deposition of C3c, C4d, and C5b-9 was detected in most patients. Levels of urinary and plasma C3a, C5a, soluble C5b-9, C4d, Bb, and C1q as well as urinary mannose-binding lectin were significantly higher in patients with PGNMID compared with healthy controls. The intensity of C3c and C4d deposition in glomeruli correlated with serum creatinine and the percentage of crescents, respectively. Furthermore, levels of urinary complement components correlated positively with serum creatinine, urinary protein excretion, percentage of crescents, and global glomerulosclerosis in kidney biopsies, whereas plasma levels of most complement components did not show a significant correlation with clinicopathologic parameters. In multivariable analysis, a higher level of urinary C4d was identified as an independent risk factor of kidney failure. CONCLUSIONS: The complement system was found to be overactivated in PGNMID, and levels of urinary complements correlated with disease severity. A higher level of urinary C4d was identified as an independent risk factor of kidney failure.