Complement C1q Research Articles

The role of gamma globulin, complement component 1q, factor B, properdin, body temperature, C-reactive protein and serum amyloid alpha to the activity and the function of the human complement system and its pathways

The complement system plays a crucial role in orchestrating the activation and regulation of inflammation within the human immune system. Three distinct activation pathways—classical, lectin, and alternative—converge to form the common lytic pathway, culminating in the formation of the membrane-attacking complex that disrupts the structure of pathogens. Dysregulated complement system activity can lead to tissue damage, autoimmune diseases, or immune deficiencies.In this study, the antimicrobial activity of human serum was investigated by using a bioluminescent microbe probe, Escherichia coli (pEGFPluxABCDEamp). This probe has previously been used to determine the antimicrobial activity of complement system and the polymorphonuclear neutrophils. In this study, blocking antibodies against key serum activators and components, including IgG, complement component 1q, factor B, and properdin, were utilized. The influence of body temperature and acute phase proteins, such as C reactive protein (CRP) and serum amyloid alpha (SAA), on the complement system was also examined.The study reveals the critical factors influencing complement system activity and pathway function. Alongside crucial factors like C1q and IgG, alternative pathway components factor B and properdin played pivotal roles. Results indicated that the alternative pathway accounted for approximately one third of the overall serum antimicrobial activity, and blocking this pathway disrupted the entire complement system. Contrary to expectations, elevated body temperature during inflammation did not enhance the antimicrobial activity of human serum.CRP demonstrated complement activation properties, but at higher physiological concentrations, it exhibited antagonistic tendencies, dampening the response. On the other hand, SAA enhanced the serum's activity.Overall, this study sheds a light on the critical factors affecting both complement system activity and pathway functionality, emphasizing the importance of a balanced immune response.

Increasing serum complement component 1q is associated with worse prognosis in advanced non-small cell lung cancer treated with immune checkpoint inhibitors: a single-center, retrospective study.

There is a lack of readily available clinical markers of non-small cell lung cancer (NSCLC) immunotherapy efficacy. Previous studies have found that overexpressed complement component 1q (C1q) promotes macrophage M2 polarization and an immunosuppressive tumor microenvironment. This study aimed to evaluate the association between serum C1q and the efficacy of immune checkpoint inhibitors (ICIs) in patients with advanced NSCLC. A total of 168 patients with advanced NSCLC who received ICIs in the Renmin Hospital of Wuhan University were included in this study. Serum C1q levels were collected before and 3 weeks after immunotherapy treatment, together with other data on clinical and demographic characteristics. The primary outcome was overall survival (OS) (months from first dose of ICIs to death, censored at date of last follow-up). Secondary outcome was progression-free survival (PFS) [defined as months from first dose of ICIs to clinical or radiographic progression by Response Evaluation Criteria in Solid Tumors version 1.1 (RECIST 1.1) or death, censored at date of last follow-up] and objective response rate (ORR) which was defined as rate of complete response (CR) or partial response (PR) at best response by RECIST 1.1. A total of 168 patients were included in this study, including 127 males (75.60%) and 41 females (24.40%). Thirty-nine patients achieved objective response (2 CR, 37 PR), and 111 patients (66.07%) had stable disease (SD) as best response. The ORR was 23.21% and the disease control rate was 89.28%. The upward trends of serum C1q levels between baseline and post-treatment were strongly associated with the shorter PFS [hazard ratio (HR) =1.554, 95% confidence interval (CI): 1.07-2.10, P=0.01] and OS (HR =1.444, 95% CI: 1.01-1.98, P=0.03). Moreover, taking the median OS 18.9 months as the cut-off of prognosis, receiver operating characteristic (ROC) analysis showed that serum baseline C1q yielded an area under the ROC curve of 0.785 (95% CI: 0.711-0.869). The optimal serum baseline C1q cut-off point to predict immunotherapy prognosis was 216.2 mg/L. These findings suggested that elevated serum C1q after ICIs treatment was related to a worse prognosis in NSCLC. Monitoring the baseline and dynamic data of C1q during hospitalization showed the potential to predict the prognosis of NSCLC patients.

Norisoboldine, a Natural Alkaloid from Lindera aggregata (Sims) Kosterm, Promotes Osteogenic Differentiation via S6K1 Signaling Pathway and Prevents Bone Loss in OVX Mice.

Norisoboldine (NOR) is a major isoquinoline alkaloid component in the traditional Chinese herbal plant Lindera aggregata (Sims) Kosterm, with previously reported anti-osteoclast differentiation and antiarthritis properties. However, the roles of NOR on osteoblasts, bone marrow mesenchymal stem cells (BMSCs), and osteoporosis in vivo have never been well established. This study investigates the ability of NOR to improve bone formation in vitro and in vivo. Osteoblasts and BMSCs are used to study the effect of NOR on osteogenic and adipogenic differentiation. It finds that NOR promotes osteogenic differentiation of osteoblasts and BMSCs, while inhibiting adipogenic differentiation of BMSCs by reducing the relative expression of peroxisome proliferator-activated receptorγ (Ppar-γ) and adiponectin, C1Q and collagen domain containing (Adipoq). Mechanistic studies show that NOR increases osteoblast differentiation through the mechanistic target of rapamycin kinase (mTOR)/ribosomal protein S6 kinase; polypeptide 1 (S6K1) pathway, and treatment with an mTOR inhibitor rapamycin blocked the NOR-induced increase in mineral accumulation. Finally, the study evaluates the therapeutic potential of NOR in a mouse model of ovariectomy (OVX)-induced bone loss. NOR prevents bone loss in both trabecular and cortical bone by increasing osteoblast number and phospho-S6K1 (p-S6K1) expression in osteoblasts. NOR effects in enhancing osteoblast-induced bone formation via S6K1 pathway, suggesting the potential of NOR in osteoporosis treatment by increasing bone formation.

#1390 Differences in the detection of anti-HLA antibodies between two single antigen bead methods in kidney transplantation patients

Abstract Background and Aims Two semiquantitative single-antigen bead (SAB) methods are currently available for the detection of anti-HLA antibodies and the estimation of their activity, depending on complement binding. However, data on accordance of the two methods on specificities detection and their reactivity evaluation are scarce. The purpose of this study is to compare the two methods in terms of anti-HLA antibodies specificities detection and of their complement binding capacity Method Sera from 97 patients with a positive Panel Reactive Antibody Test (>5%) was tested by two SAB methods, Immucor (IC) and One-lambda (OL), for class I and II anti-HLA antibody specificities and their ability to bind complement. The patients were either on a waiting list for kidney transplantation or transplanted patients tested routinely or after a clinical event. Statistical analysis of the results and comparison of the two methods followed. Results OL detected more class I positive specificities per patient [25 (9-34) vs. 18 (7-28) with IC, p < 0.001], while the two methods showed no difference in class II anti-HLA antibodies. IC detected 1608 out of 8148 and 1136 out of 7275 totally examined, positive anti-HLA class I and II antibody specificities respectively, with a median mean fluorescence intensity (MFI) of 4195 (1995-11272) and 6706 (2647-13184) respectively. Similarly, OL detected 1942/8148 and 1247/7275 positive specificities of anti-HLA class I and II antibodies respectively, with MFI 6185 (2855-12099) and 9498 (3630-17702)] respectively. In the IC method, 428/1608 [MFI 13900 (9540-17999)] and 409/1136 [MFI 11832 (7128-16531)] positive class I and II specificities, respectively, bound the C3d fraction of complement. Similarly, OL detected 485/1942 [MFI 15452 (9369-23095)] and 298/1247 [MFI 18852 (14415-24707)] class I and II specificities respectively that bound the C1q fraction of complement. In the analysis of the specificities that were positive by both methods, MFI showed a strong positive linear correlation between the two methods (r = 0.61, p < 0.001 and r = 0.54, p = 0.001 for classes I and II respectively). Accordingly, data were analyzed with Principal Components Analysis, in which negative results were also included. In the projection of the plane defined by the first two principal components the vector corresponding to the IC method for class I antibodies was congruent with the vectors of the complement binding detection methods, C3d and C1q. In contrast, the OL vector was nearly perpendicular to the other vectors, indicating the existence of non-complement-binding specificities that are not detected by IC. A similar picture was found for antibodies against HLA class II (Figure). Conclusion OL was more sensitive for detecting class I and II anti-HLA antibodies despite the fact that there was no difference in the number of class II specificities per patient. However, the additional specificities detected by OL, potentially do not bind complement. The MFI of the complement binding specificities were higher in both methods. The two methods were equivalent in detecting complement-binding class I specificities, however the C3d IC method was more sensitive in detecting complement-binding class II anti-HLA antibodies.

#2314 Circulating immune complexes concentration and complement activation in sensitized kidney transplant recipients

Abstract Background and Aims Chronic antibody mediated rejection of the kidney transplant is the most common cause of graft loss in the late posttransplant period. Crucial in this process seems to be the role of classical pathway complement activation due to the formation of immune complexes. The formation of immune complexes is a benign process of a normally functioning immune system. In kidney transplant recipients (KTRs), the production of donor specific IgG/IgM antibodies (DSAs) against donor antigens (MHC molecules, ABO blood group antigens) leads to the attachment of immune complexes to cell membranes and subsequent tissue damage through complement activation. The attachment of C1q complement fragment to the immune complexes holds an important part in this process. This study aims to determine the levels of immune complexes as well as classical pathway complement activation in sensitized kidney transplant recipients. Method In this cross-sectional study 3 groups of KTRs were compared as to the levels of immunocomplexes concentration and classical pathway complement activation. Fourteen KTRs with preformed or de novo donor specific antibodies were compared to 28 kidney transplant recipients with no DSAs and to 22 subjects with no history of chronic kidney disease (controls). We used MicroVue Complement CIC-C1q EIA that is designed to detect immunocomplexes that bind to C1q in human serum or plasma. In order to estimate total classical pathway complement activation in serum, we used MicroVue Complement CH50 Eq EIA. Results Kidney function (eGFR) of KTRs with and without DSAs was similar (53.1 ± vs. 60.8 ± ml/min/1.73 m2, p = 0.54) but lower in comparison to controls (106.4 ml/min/1.73 m2, p < 0.001). We found a significantly higher concentration of C1q-immunocomplexes in the serum of KTRs with donor specific antibodies, in comparison to controls, as well as to the group of kidney transplant recipients with no DSAs (6.78 ± 2.7 vs. 4.83 ± 1.9 vs. 5.04 ± 1.2 μgEq/ml respectively, p = 0.0078). Furthermore, there was no significant difference of C1q-immunocomplexes between kidney transplant recipients with no DSAs and controls. Finally, a significant reduction of CH50 levels was observed in KTRs with DSAs as compared to controls and KTRs with no DSAs (39.2 ± 14.9 vs. 70.6 ± 34.3 vs. 67.9 ± 39.9 U/ml respectively, p = 0.003), which suggests higher levels of classical pathway complement activation. CH50 levels in KTRs with DSAs, who were further subcategorized in those with preformed and those with de novo DSAs were similar (39.68 ± 15.6 vs 39.08 ± 16.04 U/ml). Conclusion Kidney transplant recipients with donor specific antibodies have increased serum levels of immune complexes and increased classical pathway complement activation.

Interior decorative volatile organic compounds exposure induces sleep disorders through aberrant branched chain amino acid transaminase 2 mediated glutamatergic signaling resulting from a neuroinflammatory cascade

Air pollution has been recognized as a contributing factor to sleep disorders (SD), which have been correlated with an elevated susceptibility to a variety of human diseases. Nevertheless, research has not definitively established a connection between SD and interior decorative volatile organic compounds (ID-VOCs), a significant indoor air pollutant. In this study, we employed a mouse model exposed to ID-VOCs to explore the impacts of ID-VOCs exposure on sleep patterns and the potential underlying mechanism. Of the 23 key compositions of ID-VOCs identified, aromatic hydrocarbons were found to be the most prevalent. Exposure to ID-VOCs in mice resulted in SD, characterized by prolonged wake fullness and decreased sleep during the light period. ID-VOCs exposure triggered neuroinflammatory responses in the suprachiasmatic nucleus (SCN), with microglia activation leading to the overproduction of inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin-1α (IL-1α), and complement component 1q (C1q), ultimately inducing A1 astrocytes. Consequently, the upregulation of branched chain amino acid transaminase 2 (BCAT2) in A1 astrocytes resulted in elevated extracellular glutamate and disruption of the wake-sleep transition mechanism, which might be the toxicological mechanism of SD caused by ID-VOCs.

The role of the immune system in depersonalisation disorder

Objectives Depersonalisation-derealization disorder (DPD) is a dissociative disorder that impairs cognitive function and occupational performance. Emerging evidence indicate the levels of tumour necrosis factor-α and interleukin associated with the dissociative symptoms. In this study, we aimed to explore the role of the immune system in the pathology of DPD. Methods We screened the protein expression in serum samples of 30 DPD patients and 32 healthy controls. Using a mass spectrometry-based proteomic approach, we identified differential proteins that were verified in another group of 25 DPD patients and 30 healthy controls using immune assays. Finally, we performed a correlation analysis between the expression of differential proteins and clinical symptoms of patients with DPD. Results We identified several dysregulated proteins in patients with DPD compared to HCs, including decreased levels of C-reactive protein (CRP), complement C1q subcomponent subunit B, apolipoprotein A-IV, and increased levels of alpha-1-antichymotrypsin (SERPINA3). Moreover, the expression of CRP was positively correlated with visuospatial memory and the ability to inhibit cognitive interference of DPD. The expression of SERPINA3 was positively correlated with the ability to inhibit cognitive interference and negatively correlated with the perceptual alterations of DPD. Conclusions The dysregulation of the immune system may be the underlying biological mechanism in DPD. And the expressions of CRP and SERPINA3 can be the potential predictors for the cognitive performance of DPD.

Druggability properties of a L309K mutation in the antibody CH2 domain.

In the early stages of antibody drug development, it is imperative to conduct a comprehensive assessment and enhancement of the druggability attributes of potential molecules by considering their fundamental physicochemical properties. This study specifically concentrates on the surface-exposed hydrophobic region of the candidate antibody aPDL1-WT and explores the effectiveness of the L309K mutation strategy. The resulting aPDL1-LK variant demonstrates a notable enhancement over the original antibody in addressing the issue of aggregation and formation of large molecular impurities under accelerated high-temperature conditions. The mutated molecule, aPDL1-LK, exhibits excellent physicochemical properties such as hydrophilicity, conformational stability, charge variant stability, post-translational modifications, and serum stability. In terms of biological function, aPDL1-LK maintains the same glycosylation pattern as the original antibody and shows no significant difference in affinity for antigen hPDL1 protein, CD16a-F158, CD64, CD32a-H131, and complement C1q, compared to aPDL1-WT. The L309K mutation results in an approximately twofold reduction in its affinity for CD16a-V158 and CD32a-R131. In vitro biological assays, including antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC), reveal that the L309K mutation may decrease CD16a-V158-mediated ADCC activity due to the mutation-induced decrease in ligand affinity, while not affect CD32a-R131-mediated ADCP activity. In conclusion, the L309K mutation offers a promising strategy to enhance the druggability properties of candidate antibodies.

The clinical role of combined circulating complement C1q and AIP for CAD with LDL-C level below 1.8mmol/L

BackgroundIn the past few years, circulating complement C1q involvement in atherosclerosis has garnered growing research interest in addition to the emerging recognition of the novel lipid marker named atherogenic index of plasma (AIP). Nevertheless, among patients experiencing low-density lipoprotein cholesterol (LDL-C) levels less than 1.8mmol/L, the interplay between C1q combined with the AIP for coronary artery disease (CAD) is ambiguous.MethodsPatients were stratified into a non-CAD and CAD group according to their coronary angiography. The association between C1q in conjunction with the AIP and CAD was explored using restricted cubic spline analyses and logistic regression models. To assess how it predicted, a receiver operating characteristic analysis was undertaken.ResultsA total of 7270 patients comprised 1476 non-CAD patients and 5794 patients diagnosed with CAD were analyzed. A comparison of the two groups showed that the C1q levels were notably higher compared to the CAD group, while AIP exhibited an inverse trend. Across quartiles of C1q, the AIP demonstrated a decline with increasing C1q levels, and significant differences were observed between the groups. A correlation analysis underscored a notable negative correlation between the two variables. Univariate and multivariate logistic regression analyses revealed significant associations between CAD and the C1q quartile groups/AIP. Furthermore, compared with the Q4 group, a decrease in the C1q levels corresponded to an escalation in CAD risk, with the odds ratio rising from 1.661 to 2.314.ConclusionsIn conclusion, there appears to be a notable positive correlation between the combination of C1q with the AIP and CAD.

DNA Nanostructure-Templated Antibody Complexes Provide Insights into the Geometric Requirements of Human Complement Cascade Activation.

The classical complement pathway is activated by antigen-bound IgG antibodies. Monomeric IgG must oligomerize to activate complement via the hexameric C1q complex, and hexamerizing mutants of IgG appear as promising therapeutic candidates. However, structural data have shown that it is not necessary to bind all six C1q arms to initiate complement, revealing a symmetry mismatch between C1 and the hexameric IgG complex that has not been adequately explained. Here, we use DNA nanotechnology to produce specific nanostructures to template antigens and thereby spatially control IgG valency. These DNA-nanotemplated IgG complexes can activate complement on cell-mimetic lipid membranes, which enabled us to determine the effect of IgG valency on complement activation without the requirement to mutate antibodies. We investigated this using biophysical assays together with 3D cryo-electron tomography. Our data revealed the importance of interantigen distance on antibody-mediated complement activation, and that the cleavage of complement component C4 by the C1 complex is proportional to the number of ideally spaced antigens. Increased IgG valency also translated to better terminal pathway activation and membrane attack complex formation. Together, these data provide insights into how nanopatterning antigen-antibody complexes influence the activation of the C1 complex and suggest routes to modulate complement activation by antibody engineering. Furthermore, to our knowledge, this is the first time DNA nanotechnology has been used to study the activation of the complement system.

The relationship between complement C1q and coronary plaque vulnerability based on optical coherence tomography analysis

To determine the association between complement C1q and vulnerable plaque morphology among coronary artery disease (CAD) patients. We conducted a retrospective observational study of 221 CAD patients admitted to The Second Affiliated Hospital of Xi’an Jiaotong University. Intravascular optical coherence tomography was utilized to describe the culprit plaques’ morphology. Using logistic regression analysis to explore the correlation between C1q and vulnerable plaques, and receiver operator characteristic (ROC) analysis assess the predictive accuracy. As reported, the complement C1q level was lower in ACS patients than CCS patients (18.25 ± 3.88 vs. 19.18 ± 4.25, P = 0.045). The low complement-C1q-level group was more prone to develop vulnerable plaques. In lipid-rich plaques, the complement C1q level was positively correlated with the thickness of fibrous cap (r = 0.480, P = 0.041). Univariate and multivariate logistic regression analyses suggested that complement C1q could be an independent contributor to plaques’ vulnerability. For plaque rupture, erosion, thrombus, and cholesterol crystals, the areas under the ROC curve of complement C1q level were 0.873, 0.816, 0.785, and 0.837, respectively (P < 0.05 for all). In CAD patients, the complement C1q could be a valuable indicator of plaque vulnerability.

Complement C1q is involved in the activation of membrane attack complexes, regulation of bacterial infectious inflammation, and apoptosis through overexpression in primary cells of silver pomfret (Pampus argenteus) in vitro

The complement system is pivotal in innate immune defense, with Complement 1qb (C1qb) playing a key role in recognizing immune complexes and initiating the classical pathway. In this research, we cloned the full-length cDNA of silver pomfret (Pampus argenteus) c1qb and demonstrated its role in mediating defense responses against Nocardia seriolae (N. seriolae) infection, which notably causes significant economic losses in the aquaculture industry. Our investigation revealed that N. seriolae infection led to tissue damage in fish bodies, as observed in tissue sections. Subsequent analysis of differential genes (DEGs) in the transcriptome highlighted genes linked to apoptosis and inflammation. Through experiments involving overexpression and interference of c1qb in vitro, we confirmed that c1qb could suppress N. seriolae-induced apoptosis and inflammation. Moreover, overexpression of c1qb hindered N. seriolae invasion, and the purified and replicated C1qb protein displayed antimicrobial properties. Additionally, our study unveiled that overexpression of c1qb might stimulate the expression of membrane attack complexes (MAC), potentially enhancing opsonization and antibacterial effects. In conclusion, our findings offer valuable insights into the immune antibacterial mechanisms of c1qb and contribute to the development of strategies for controlling N. seriolae.

Effect of inflammatory response on joint function after hip fracture in elderly patients: A clinical study

BACKGROUND Excellent hip joint function facilitates limb recovery and improves the quality of survival. This study aimed to investigate the potential risk factors affecting postoperative joint functional activity and outcomes in elderly hip fractures patients and to provide evidence for patient rehabilitation and clinical management. AIM To explore the relationship between inflammatory factors and hip function and the interaction between inflammation and health after hip fracture in elderly patients. METHODS The elderly patients who had hip fracture surgery at our hospital between January 1, 2021, and December 31, 2022 were chosen for this retrospective clinical investigation. Patients with excellent and fair postoperative hip function had their clinical information and characteristics gathered and compared. Age, gender, fracture site, surgical technique, laboratory indices, and other variables that could have an impact on postoperative joint function were all included in a univariate study. To further identify independent risk factors affecting postoperative joint function in hip fractures, risk factors that showed statistical significance in the univariate analysis were then included in a multiple logistic regression analysis. In addition to this, we also compared other outcome variables such as visual analogue scale and length of hospital stay between the two groups. RESULTS A total of 119 elderly patients with hip fractures were included in this study, of whom 37 were male and 82 were female. The results of univariate logistic regression analysis after excluding the interaction of various factors showed that there was a statistically significant difference in interleukin (IL)-6, IL-8, IL-10, C-reactive protein (CRP), and complement C1q (C1q) between the fair and excellent joint function groups (P &lt; 0.05). The results of multiple logistic regression analysis showed that IL-6 &gt; 20 pg/mL [(Odds ratio (OR) 3.070, 95%CI: 1.243-7.579], IL-8 &gt; 21.4 pg/ mL (OR 3.827, 95%CI: 1.498-9.773), CRP &gt; 10 mg/L (OR 2.142, 95%CI: 1.020-4.498) and C1q &gt; 233 mg/L (OR 2.339, 95%CI: 1.094-5.004) were independent risk factors for poor joint function after hip fracture surgery (all P &lt; 0.05). CONCLUSION After hip fractures in older patients, inflammatory variables are risk factors for fair joint function; therefore, early intervention to address these markers is essential to enhance joint function and avoid consequences.

Proteomic profiling of circulating β-thalassaemia/haemoglobin E extra-cellular vesicles reveals that association with immunoglobulin induces membrane vesiculation.

Splenectomised β-thalassaemia/haemoglobin E (HbE) patients have increased levels of circulating microparticles or medium extra-cellular vesicles (mEVs). The splenectomised mEVs play important roles in thromboembolic complications in patients since they can induce platelet activation and endothelial cell dysfunction. However, a comprehensive understanding of the mechanism of mEV generation in thalassaemia disease has still not been reached. Thalassaemic mEVs are hypothesised to be generated from cellular oxidative stress in red blood cells (RBCs) and platelets. Therefore, a proteomic analysis of mEVs from splenectomised and non-splenectomised β-thalassaemia/HbE patients was performed by liquid chromatography with tandem mass spectrometry. A total of 171 proteins were identified among mEVs. Interestingly, 72 proteins were uniquely found in splenectomised mEVs including immunoglobulin subunits and cytoskeleton proteins. Immunoglobulin G (IgG)-bearing mEVs in splenectomised patients were significantly increased. Furthermore, complement C1q was detected in both mEVs with IgG binding and mEVs without IgG binding. Interestingly, the percentage of mEVs generated from RBCs with IgG binding was approximately 15-20 times higher than the percentage of RBCs binding with IgG. This suggested that the vesiculation of thalassaemia mEVs could be a mechanism of RBCs to eliminate membrane patches harbouring immune complex and may consequently prevent cells from phagocytosis and lysis.

Complement C1q-mediated microglial synaptic elimination by enhancing desialylation underlies sevoflurane-induced developmental neurotoxicity

BackgroundRepeated neonatal sevoflurane exposures led to neurocognitive disorders in young mice. We aimed to assess the role of microglia and complement C1q in sevoflurane-induced neurotoxicity and explore the underlying mechanisms.MethodsNeonatal mice were treated with sevoflurane on postnatal days 6, 8, and 10, and the Morris water maze was performed to assess cognitive functions. For mechanistic explorations, mice were treated with minocycline, C1q-antibody ANX005, and sialidase-inhibitor N-acetyl-2,3-dehydro-2-deoxyneuraminic acid (NADNA) before sevoflurane exposures. Western blotting, RT-qPCR, Golgi staining, 3D reconstruction and engulfment analysis, immunofluorescence, and microglial morphology analysis were performed. In vitro experiments were conducted in microglial cell line BV2 cells.ResultsRepeated neonatal sevoflurane exposures resulted in deficiencies in learning and cognition of young mice, accompanied by microglial activation and synapse loss. Sevoflurane enhanced microglia-mediated synapse elimination through C1q binding to synapses. Inhibition of microglial activation and phagocytosis with minocycline significantly reduced the loss of synapses. We further revealed the involvement of neuronal sialic acids in this process. The enhanced activity of sialidase by sevoflurane led to the loss of sialic acids, which facilitated C1q binding to synapses. Inhibition of C1q with ANX005 or inhibition of sialidase with NADNA significantly rescued microglia-mediated synapse loss and improved neurocognitive function. Sevoflurane enhanced the engulfment of BV2 cells, which was reversed by ANX005.ConclusionsOur findings demonstrated that C1q-mediated microglial synaptic elimination by enhancing desialylation contributed to sevoflurane-induced developmental neurotoxicity. Inhibition of C1q or sialidase may be a potential therapeutic strategy for this neurotoxicity.

C1q enables influenza hemagglutinin stem binding antibodies to block viral attachment and broadens the antibody escape repertoire.

Antigenic drift, the gradual accumulation of amino acid substitutions in the influenza virus hemagglutinin (HA) receptor protein, enables viral immune evasion. Antibodies (Abs) specific for the drift-resistant HA stem region are a promising universal influenza vaccine target. Although anti-stem Abs are not believed to block viral attachment, here we show that complement component 1q (C1q), a 460-kilodalton protein with six Ab Fc-binding domains, confers attachment inhibition to anti-stem Abs and enhances their fusion and neuraminidase inhibition. As a result, virus neutralization activity in vitro is boosted up to 30-fold, and in vivo protection from influenza PR8 infection in mice is enhanced. These effects reflect increased steric hindrance and not increased Ab avidity. C1q greatly expands the anti-stem Ab viral escape repertoire to include residues throughout the HA, some of which cause antigenic alterations in the globular region or modulate HA receptor avidity. We also show that C1q enhances the neutralization activity of non-receptor binding domain anti-SARS-CoV-2 spike Abs, an effect dependent on spike density on the virion surface. These findings demonstrate that C1q can greatly expand Ab function and thereby contribute to viral evolution and immune escape.

Abstract 3190: Twin Fc-immune cell engager (ICE): A novel antibody platform for cancer immunotherapy with enhanced avidity for Fcγ receptors and complement

Abstract Immunoglobulin G (IgG)-based mAbs have become a dominant class of cancer immunotherapy due to their excellent drug-like properties, long half-life, and generally safe profiles. Typical IgG-based therapeutic mAbs have strong binding to target antigens owing to the valency effect of two antigen-binding fragments (Fabs). However, the single crystallizable fragment (Fc) lacks the valency effect to engage immune effector elements. As Fcγ receptors (FcγR) such as FcγRIIa and FcγRIIIa and C1q complement component have low affinity for Fc, IgG-based mAbs require high antibody opsonization density to exhibit avidity for these low affinity Fcγ receptors and C1q. This may also account for the suboptimal efficacy of IgG-based mAbs against tumors with less abundant antigen. To enhance effector functions of IgG1 by overcoming such limitation, we devised a novel platform, Twin Fc-ICE, in which a single Fab region is attached by two Fc regions which allow for high avidity binding to Fcγ receptors and C1q. The two Fc regions of Twin Fc-ICE are arranged in parallel, not in tandem, to adopt natural topologies of multiple Fc regions presented by IgG1-opsonized targets. Supporting the avidity effect of Twin Fc-ICE for FcγR binding, we showed that CTN001, a Twin Fc-ICE with trastuzumab Fab, have markedly increased binding to FcγRs including FcγRI, FcγRIIa and FcγRIIIa compared with trastuzumab. The avid FcγR binding was further demonstrated by flow cytometry analysis of human blood which revealed significantly increased binding of CTN001 relative to trastuzumab for immune cells expressing activating FcγRs such as natural killer (NK) cells, monocytes, and dendritic cells (DCs). The monovalent antigen binding of Twin Fc-ICE requires Fab with high affinity. However, provided that high affinity is achieved, the monovalent Fab adds to the two Fc regions to maximize the Fc load on cancer cells expressing low levels of antigen. Indeed, flow cytometry analysis revealed that treatment of CTN001 alone results in higher Fc load on HER2-expressing cancer cells than combination of two anti-HER2 IgG1 mAbs, trastuzumab and pertuzumab. In consistent with increased Fc load on target cells and avid FcγR binding, CTN001 induces enhanced effector functions such as CDC, ADCC and ADCP against HER2-expressing cancer cells compared with trastuzumab, which will be presented in a separate poster. CTN001 exhibits comparable thermal stability with trastuzumab and can be readily purified to &amp;gt;95% purity with typical purification platforms. The serum half-life of CTN001 at 10 mg/kg intravenous dose in rats is found non-inferior to that of trastuzumab (~12 days versus ~7 days). Taken together, Twin Fc-ICE provides an optimized platform with drug-like properties, which leverages avidity effects of Fc to potentiate therapeutic efficacy of antibody. Citation Format: Eun Shik Choi, Hyunkyu Park, Jiseon Bae, Ki-June Lee, Eunmi Kim, Sujin Yoon, Deokjae Lee, Gi-Hyeok Yang. Twin Fc-immune cell engager (ICE): A novel antibody platform for cancer immunotherapy with enhanced avidity for Fcγ receptors and complement [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3190.

Abstract 7461: Obesity promotes breast cancer progression via complement 1q

Abstract Obesity markedly impacts the onset and progression of various tumors. Yet, the mechanisms by which obesity-driven remodeling in macrophages modulates tumor progression are not yet fully elucidated. Using single-cell RNA sequencing and in vivo syngeneic murine models, we identified an enriched subset of macrophages expressing high levels of C1q complex genes in the adipose tissue of obese mice. Intriguingly, these C1q-positive macrophages are also prevalent within the tumor microenvironment. Our data suggest that obesity induces cell apoptosis in both adipocytes and tumor cells, and that macrophages engulf these apoptotic cells via a C1q-dependent pathway, thus promoting immunosuppression. Moreover, C1q deficiency in mice impairs tumor growth. Notably, C1q is upregulated in human breast cancer and is associated with poor prognosis. Our results suggest that targeting C1q could be an effective method for breast cancer treatment. Citation Format: Tao Zhang, Shimeng Liu, Myles Brown. Obesity promotes breast cancer progression via complement 1q [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7461.

A single C1q A chain promotes leukocytes phagocytosis/chemotaxis and mediates bacterial clearance in Japanese flounder Paralichthys olivaceus

Complement component 1q (C1q) is the first component involved in the activation of complement classical pathway and acts as a linker of the innate and adaptive immune system. The C1q is a macromolecular containing three chains i.e. C1q A, C1q B, and C1q C. In teleost, the biological functions of C1q A chain is still unclear. In this study, we investigated the immunological properties of a C1q A chain from Japanese flounder (Paralichthys olivaceus) (PoC1qA). PoC1qA containing the conserved CLR and C1q domain, shares 45.4%–78.6% identities with that of other teleost and mammals. PoC1qA expression pattern was constitutive in tissues, with higher expression in spleen and liver. Besides, the PoC1qA expression was notably regulated by pathogenic invasion. In vitro the recombinant PoC1qA (rPoC1qA) could directly bind a variety of bacteria, and affect bacterial growth and survival. Moreover, PoC1qA could influence the hemolysis and bactericidal activity of fish serum. Additionally, rPoC1qA could interact with the membrane of peripheral blood leucocytes (PBLs), promoting the PBLs phagocytosis and chemotaxis, and enhancing the activation of PBLs. Consistently, in vivo rPoC1qA could restrain the bacterial replication in immune tissues. To sum up, our observations added the first evidence that an A chain of teleost C1q plays vital roles in mediating PBLs activity and antibacterial immunity.

A novel teleost C1q C chain (PoC1qC) participates in complement and leukocytes antibacterial immunity

Complement C1q composed of C1q A, B, C chains is the initial vital protein of classical complement pathway, playing an essential role in innate immune response. Whereas in teleost, the immune roles of C1q C chain are largely scarce. In this research, we studied the complement/peripheral blood leukocytes (PBLs) activation and antibacterial potentials mediated by Japanese flounder (Paralichthys olivaceus) C1q C chain (PoC1qC). The PoC1qC shares 56.7%–87.6% identities with that of teleost and mammals. The expression of PoC1qC can be detected in various organs, notably up-regulated by V. harveyi stimulation, and down-regulated by E. tarda infection. The recombinant PoC1qC (rPoC1qC) could directly bind to different bacteria and exhibit antibacterial potential. Consistently, rPoC1qC and anti-rPoC1qC antibody could remarkably enhance or suppress the complement hemolytic and bactericidal activity, respectively. Furthermore, rPoC1qC could directly interact with PBLs, resulting in the activation of proliferation, chemotaxis, and phagocytosis of PBLs, and the generation of ROS/acid phosphatase activity of PBLs. Lastly, we found in vivo rPoC1qC could promote the host to resistant microbial invasion. Taken together, our findings provide the first evidence that PoC1qC played pivotal roles in complement and PBLs activation and antibacterial activity.