CSF complement proteins are elevated in prodromal to moderate Alzheimer's disease patients and are not altered by the anti-tau antibody semorinemab.

INTRODUCTION: Growing evidence suggests a role for neuroinflammation in Alzheimer's disease (AD). We investigated complement pathway activity in AD patient cerebrospinal fluid (CSF) and evaluated its modulation by the anti-tau antibody semorinemab. METHODS: Immunoassays were applied to measure CSF complement proteins C4, factor B (FB), C3 and their cleavage fragments C4a, C3a, and factor Bb (Bb) in AD patients and a separate cognitively unimpaired (CU) cohort. RESULTS: All measured CSF complement proteins were increased in AD versus CU subjects, with C4a displaying the most robust increase. Finally, semorinemab did not have a significant pharmacodynamic effect on CSF complement proteins. DISCUSSION: Elevated levels of CSF C4a, C4, C3a, C3, Bb, and FB are consistent with complement activation in AD brains. Despite showing a reduction in CSF soluble tau species, semorinemab did not impact complement protein levels or activity. Further studies are needed to determine the value of complement proteins as neuroinflammation biomarkers in AD. HIGHLIGHTS: Cerebrospinal fluid (CSF) complement proteins C4a, C3a, Bb, C4, C3, and factor B levels were increased in Alzheimer's disease (AD) patients compared to a separate cognitively unimpaired (CU) cohort. Baseline CSF complement protein levels were correlated with neuro-axonal degeneration and glial activation biomarkers in AD patients. The investigational anti-tau antibody semorinemab did not impact CSF complement protein levels or activity relative to the placebo arm.

Protein Network Alterations in G-CSF Treated Severe Congenital Neutropenia Patients and Beneficial Effects of Oral Health Intervention.

PURPOSE: Severe congenital neutropenia (SCN) is a raredisorder characterized by diminished neutrophil levels. Despite granulocytecolony-stimulating factor (G-CSF) treatment, SCN patients remain still prone tosevere infections, including periodontal disease-a significant oral healthrisk. This study investigates the host proteome and metaproteome in saliva andgingival crevicular fluid (GCF) of G-CSF-treated patients. EXPERIMENTAL DESIGN: We used label-free quantitative proteomics on saliva and GCF samples from SCN patients before (n = 10, mean age: 10.7 ± 6.6 years) and after a 6-month oral hygiene intervention (n = 9,mean age: 11.6 ± 5.27 years), and from 12 healthy controls. RESULTS: We quantified 894 proteins in saliva (648 human,246 bacterial) and 756 proteins in GCF (493 human, 263 bacterial). Predominant bacterial genera included Streptococcus, Veillonella, Selenomonas, Corynebacterium, Porphyromonas, and Prevotella. SCN patients showed reduced antimicrobial peptides (AMPs) and elevated complement proteins compared tohealthy controls. Oral hygiene intervention improved oral epithelial conditionsand reduced both AMPs and complement proteins. CONCLUSIONS AND CLINICAL RELEVANCE: SCN patients have aunique proteomic profile with reduced AMPs and increased complement proteins, contributing to infection susceptibility. Oral hygiene intervention not onlyimproved oral health in SCN patients but also offers potential overall therapeuticbenefits.

Contributions of Lipid-Related Metabolites and Complement Proteins to Early and Intermediate Age-Related Macular Degeneration.

Objective: Our objective was to determine the effects of lipids and complement proteins on early and intermediate age-related macular degeneration (AMD) stages using machine learning models by integrating metabolomics and proteomic data. Design: Nested case-control study. Subjects and Controls: The analyses were performed in a subset of the Singapore Indian Chinese Cohort (SICC) Eye Study. Among the 6753 participants, we randomly selected 155 Indian and 155 Chinese cases of AMD and matched them with 310 controls on age, sex, and ethnicity. Methods: We measured 35 complement proteins and 56 lipids using mass spectrometry and nuclear magnetic resonance, respectively. We first selected the most contributing lipids and complement proteins to early and intermediate AMD using random forest models. Then, we estimated their effects using a multinomial model adjusted for potential confounders. Main Outcome Measures: Age-related macular degeneration was classified using the Beckman classification system. Results: Among the 310 individuals with AMD, 166 (53.5%) had early AMD, and 144 (46.5%) had intermediate AMD. First, high-density lipoprotein (HDL) particle diameter was positively associated with both early and intermediate AMD (odds ratio [OR]early = 1.69; 95% confidence interval [CI],1.11-2.55 and ORintermediate = 1.72; 95% CI, 1.11-2.66 per 1-standard deviation increase in HDL diameter). Second, complement protein 2 (C2), complement C1 inhibitor (IC1), complement protein 6 (C6), complement protein 1QC (C1QC) and complement factor H-related protein 1 (FHR1), were associated with AMD. C2 was positively associated with both early and intermediate AMD (ORearly = 1.58; 95% CI, 1.08-2.30 and ORintermediate = 1.56; 95% CI, 1.04-2.34). C6 was positively (ORearly = 1.41; 95% CI, 1.03-1.93) associated with early AMD. However, IC1 was negatively associated with early AMD (ORearly = 0.62; 95% CI, 0.38-0.99), whereas C1QC (ORintermediate = 0.63; 95% CI, 0.42-0.93) and FHR1 (ORintermediate = 0.73; 95% CI, 0.54-0.98) were both negatively associated with intermediate AMD. Conclusions: Although both HDL diameter and C2 levels show associations with both early and intermediate AMD, dysregulations of IC1, C6, C1QC, and FHR1 are only observed at specific stages of AMD. These findings underscore the complexity of complement system dysregulation in AMD, which appears to vary depending on the disease severity. Financial Disclosures: The authors have no proprietary or commercial interest in any materials discussed in this article.

Beneficial versus Detrimental Effects of Complement-Microglial Interactions in Alzheimer's Disease.

Research indicates that brain-region-specific synapse loss and dysfunction are early hallmarks and stronger neurobiological correlates of cognitive decline in Alzheimer's disease (AD) than amyloid plaque and neurofibrillary tangle counts or neuronal loss. Even though the precise mechanisms underlying increased synaptic pruning in AD are still unknown, it has been confirmed that dysregulation of the balance between complement activation and inhibition is a crucial driver of its pathology. The complement includes three distinct activation mechanisms, with the activation products C3a and C5a, potent inflammatory effectors, and a membrane attack complex (MAC) leading to cell lysis. Besides pro-inflammatory cytokines, the dysregulated complement proteins released by activated microglia bind to amyloid ß at the synaptic regions and cause the microglia to engulf the synapses. Additionally, research indicating that microglia-removed synapses are not always degenerating and that suppression of synaptic engulfment can repair cognitive deficits points to an essential opportunity for intervention that can prevent the loss of intact synapses. In this study, we focus on the latest research on the role and mechanisms of complement-mediated microglial synaptic pruning at different stages of AD to find the right targets that could interfere with complement dysregulation and be relevant for therapeutic intervention at the early stages of the disease.

Decay-Accelerating Factor Differentially Associates With Complement-Mediated Damage in Synovium After Meniscus Tear as Compared to Anterior Cruciate Ligament Injury.

We have reported that anterior cruciate ligament (ACL) injury leads to the differential dysregulation of the complement system in the synovium as compared to meniscus tear (MT) and proposed this as a mechanism for a greater post-injury prevalence of post traumatic osteoarthritis (PTOA). To explore additional roles of complement proteins and regulators, we determined the presence of decay-accelerating factor (DAF), C5b, and membrane attack complexes (MACs, C5b-9) in discarded surgical synovial tissue (DSST) collected during arthroscopic ACL reconstructive surgery, MT-related meniscectomy, osteoarthritis (OA)-related knee replacement surgery and normal controls. Multiplexed immunohistochemistry was used to detect and quantify complement proteins. To explore the involvement of body mass index (BMI), after these 2 injuries, we examined correlations among DAF, C5b, MAC and BMI. Using these approaches, we found that synovial cells after ACL injury expressed a significantly lower level of DAF as compared to MT (p<0.049). In contrast, C5b staining synovial cells were significantly higher after ACL injury (p<0.0009) and in OA DSST (p<0.039) compared to MT. Interestingly, there were significantly positive correlations between DAF & C5b (r=0.75, p<0.018) and DAF & C5b (r=0.64 p<0.022) after ACL injury and MT, respectively. The data support that DAF, which should normally dampen C5b deposition due to its regulatory activities on C3/C5 convertases, does not appear to exhibit that function in inflamed synovia following either ACL injury or MT. Ineffective DAF regulation may be an additional mechanism by which relatively uncontrolled complement activation damages tissue in these injury states.

Complement Proteins in Serum Astrocyte-Derived Exosomes Are Associated with Poststroke Cognitive Impairment in Type 2 Diabetes Mellitus Patients.

Background: The complement system plays crucial roles in cognitive impairment and acute ischemic stroke (AIS). High levels of complement proteins in plasma astrocyte-derived exosomes (ADEs) were proven to be associated with Alzheimer's disease. We aimed to investigate the relationship of complement proteins in serum ADEs with poststroke cognitive impairment in type 2 diabetes mellitus (T2DM) patients. Methods: This study analyzed 197 T2DM patients who suffered AIS. The Beijing version of the Montreal Cognitive Assessment (MoCA) was used to assess cognitive function. Complement proteins in serum ADEs were quantified using ELISA kits. Results: Mediation analyses showed that C5b-9 and C3b in serum ADEs partially mediate the impact of obstructive sleep apnea (OSA), depression, small vessel disease (SVD), and infarct volume on cognitive function at the acute phase of AIS in T2DM patients. After adjusting for age, sex, time, and interaction between time and complement proteins in serum ADEs, the mixed linear regression showed that C3b and complement protein Factor B in serum ADEs were associated with MoCA scores at three-, six-, and twelve-months after AIS in T2DM patients. Conclusions: Our study suggested that the impact of OSA, depression, SVD, and infarct volume on cognitive impairment in the acute stage of AIS may partially mediate through the complement proteins in serum ADEs. Additionally, the complement proteins in serum ADEs at the acute phase of AIS associated with MoCA scores at three-, six-, twelve months after AIS in T2DM patients.REGISTRATION: URL: http://www.chictr.org.cn/,ChiCTR1900021544.

Humoral complementomics - exploration of noninvasive complement biomarkers as predictors of renal cancer progression.

Despite the progress of anti-cancer treatment, the prognosis of many patients with solid tumors is still dismal. Reliable noninvasive biomarkers are needed to predict patient survival and therapy response. Here, we propose a Humoral Complementomics approach: a work-up of assays to comprehensively evaluate complement proteins, activation fragments, and autoantibodies targeting complement proteins in plasma, which we correlated with the intratumoral complement activation, and/or local production, focusing on localized and metastatic clear cell renal cell carcinoma (ccRCC). In two prospective ccRCC cohorts, plasma C2, C5, Factor D and properdin were elevated compared to healthy controls, reflecting an inflammatory phenotype that correlated with plasma calprotectin levels but did not associate with CRP or with patient prognosis. Conversely, autoantibodies against the complement C3 and the reduced form of FH (a tumor neo-epitope reported in lung cancer) correlated with a favorable outcome. Our findings pointed to a specific group of patients with elevated plasma C4d and C1s-C1INH complexes, indicating the initiation of the classical pathway, along with elevated Ba and Bb, indicating alternative pathway activation. Boostrapped Lasso regularized Cox regression revealed that the most predictive complement biomarkers were elevated plasma C4d and Bb levels at the time of surgery, which correlated with poor prognosis. In conclusion, we propose Humoral Complementomics as an unbiased approach to study the global state of the complement system in any pathological plasma sample and disease context. Its implementation for ccRCC revealed that elevated C4d and Bb in plasma are promising prognostic biomarkers, correlating with shorter progression-free survival.

Botulinum Neurotoxin Induces Neurotoxic Microglia Mediated by Exogenous Inflammatory Responses.

Botulinum neurotoxin serotype A (BoNT/A) is widely used in therapeutics and cosmetics. The effects of multi-dosed BoNT/A treatment are well documented on the peripheral nervous system (PNS), but much less is known on the central nervous system (CNS). Here, the mechanism of multi-dosed BoNT/A leading to CNS neurodegeneration is explored by using the 3D human neuron-glia model. BoNT/A treatment reduces acetylcholine, triggers astrocytic transforming growth factor beta, and upregulates C1q, C3, and C5 expression, inducing microglial proinflammation. The disintegration of the neuronal microtubules is escorted by microglial nitric oxide, interleukin 1ß, tumor necrosis factor α, and interleukin 8. The microglial proinflammation eventually causes synaptic impairment, phosphorylated tau (pTau) aggregation, and the loss of the BoNT/A-treated neurons. Taking a more holistic approach, the model will allow to assess therapeutics for the CNS neurodegeneration under the prolonged use of BoNT/A.

Complement System Proteins in the Human Aqueous Humor and Their Association with Primary Open-Angle Glaucoma.

This study discovers the complement protein profile in the aqueous humor (AH) of human subjects and investigates its association with primary open-angle glaucoma (POAG) pathogenesis. Among the 32 complement proteins identified, 22 were highly abundant and detected in more than 50% of AH samples. The most predominant active complement proteins in the AH are C3, C4B, C4A, CFB, CFD, and C9. Additionally, the most prevalent complement regulators and receptors include CLU, SERPING1, F2, CFH, CFI, and VTN. Significant alterations in complement proteins were observed in individuals with POAG compared to those with cataracts. Specifically, complement protein F2 was upregulated, while C8G, C6, and CFH were downregulated in POAG samples. Stratification of the samples by race and sex revealed distinct alterations of complement proteins in patients with POAG. In the African American cohort, five complement proteins (C4A, C4B, F2, C7, and C3) were upregulated in POAG compared to cataract patients. In the Caucasian cohort, eight complement proteins (C3, SERPING1, CFI, CLU, CFHR1, C8G, C6, and CFH) were downregulated in the POAG samples compared to the cataract samples. Within the male cohort, three complement proteins (CLU, C6, and CFH) were downregulated in POAG patients compared to those with cataracts. Whereas, within the female cohort, two complement proteins (C4B and F2) were upregulated and one (C8G) downregulated in the POAG samples when compared to cataracts. Discerning these changes in the AH complement protein profile will assist in the development of tailored therapies to modulate the complement system for managing ocular disorders. These insights may also lead to novel biomarkers for diagnosing and monitoring disease progression.

Harnessing the immunomodulatory effects of exercise to enhance the efficacy of monoclonal antibody therapies against B-cell haematological cancers: a narrative review.

Therapeutic monoclonal antibodies (mAbs) are standard care for many B-cell haematological cancers. The modes of action for these mAbs include: induction of cancer cell lysis by activating Fcγ-receptors on innate immune cells; opsonising target cells for antibody-dependent cellular cytotoxicity or phagocytosis, and/or triggering the classical complement pathway; the simultaneous binding of cancer cells with T-cells to create an immune synapse and activate perforin-mediated T-cell cytotoxicity against cancer cells; blockade of immune checkpoints to facilitate T-cell cytotoxicity against immunogenic cancer cell clones; and direct delivery of cytotoxic agents via internalisation of mAbs by target cells. While treatment regimens comprising mAb therapy can lead to durable anti-cancer responses, disease relapse is common due to failure of mAb therapy to eradicate minimal residual disease. Factors that limit mAb efficacy include: suboptimal effector cell frequencies, overt immune exhaustion and/or immune anergy, and survival of diffusely spread tumour cells in different stromal niches. In this review, we discuss how immunomodulatory changes arising from exposure to structured bouts of acute exercise might improve mAb treatment efficacy by augmenting (i) antibody-dependent cellular cytotoxicity, (ii) antibody-dependent cellular phagocytosis, (iii) complement-dependent cytotoxicity, (iv) T-cell cytotoxicity, and (v) direct delivery of cytotoxic agents.

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.

Complement proteins in serum astrocyte-derived exosomes are associated with mild cognitive impairment in type 1 diabetes mellitus patients.

BACKGROUND: The complement system plays a crucial role in cognitive impairment. The aim of this study is to investigate the correlation between the complement proteins levels in serum astrocyte-derived exosomes (ADEs) and mild cognitive impairment (MCI) in type 1 diabetes mellitus (T1DM) patients. METHODS: In this cross-sectional study, the patients with immune-mediated T1DM were enrolled. Healthy subjects matched for age and sex with T1DM patients were selected as controls. The cognitive function was evaluated by a Beijing version of the Montreal Cognitive Assessment (MoCA) questionnaire. The complement proteins including C5b-9, C3b and Factor B in serum ADEs were measured by ELISA kits. RESULTS: This study recruited 55 subjects immune-mediated T1DM patients without dementia, including 31 T1DM patients with MCI, 24 T1DM patients without MCI. 33 healthy subjects were enrolled as controls. The results showed higher complement proteins including C5b-9, C3b and Factor B levels in ADEs from T1DM patients with MCI than those in the controls (P < 0.001, P < 0.001, P = 0.006) and T1DM patients without MCI (P = 0.02, P = 0.02, P = 0.03). The C5b-9 levels in ADEs were independently associated with MCI in T1DM patients(OR: 1.20, 95% CI: 1.00-1.44, P = 0.04). The C5b-9 levels in ADEs were significantly correlated with global cognitive scores (ß = -0.360, P＜0.001) and visuo-executive (ß = -0.132, P＜0.001), language(ß = -0.036, P = 0.026) and delayed recall score (ß = -0.090,P = 0.007). There was no correlation between the C5b-9 levels in ADEs and the fasting glucose, HbA1c, fasting c-peptide and GAD65 antibody in T1DM patients. Furthermore, the C5b-9, C3b and Factor B levels in ADEs exhibited a fair combined diagnostic value for MCI, with an area under the curve of 0.76 (95% CI: 0.63-0.88, P = 0.001). CONCLUSION: The elevated C5b-9 levels in ADEswere significantly associated with theMCI in T1DM patients. The C5b-9 in ADEs may be used as a marker of MCI in T1DM patients.

Methods to Analyze the Contribution of Complement Evasion Factor (CEF) to Streptococcus pyogenes Virulence.

Group A Streptococcus (GAS, Streptococcus pyogenes) is an exclusively human pathogen that causes a range of diseases, including pharyngitis, tonsillitis, impetigo, erysipelas, necrotizing fasciitis, and toxic shock syndrome. Post-streptococcal sequelae include acute rheumatic fever and rheumatic heart disease. The bacterium produces a large arsenal of virulence factors that contribute to host tissue adhesion/colonization, bacterial spread, and host immune evasion. Immune evasion factors include proteins that interfere with complement, a system of plasma proteins that are activated by pathogens resulting in a variety of reactions on the surface of the pathogen. This leads to the activation of active components with a variety of effector functions, such as cell lysis, opsonization, and chemotaxis of phagocytes to the site of infection. We have recently identified a novel "complement evasion factor" (CEF) in S. pyogenes. CEF directly interacts with complement proteins C1r, C1s, C3, and C5, interrupts all three complement pathways, and prevents opsonization of the bacterial surface with C3b. We here present methods used to analyze the complement interference of CEF.

Prognostic Significance of the Coagulation and Complement Systems in Critical COVID-19 Infection.

Infection with the SARS-CoV-2 virus (COVID-19 disease) can cause a wide range of clinical situations - from an asymptomatic state to fatal outcomes. In cases of serious clinical manifestations, the underlying mechanisms involve a number of immune cells and stromal cells as well as their products such as pro-inflammatory interleukin-6 and tumour necrosis factor-alpha that ultimately cause the cytokine storm. The situation of overproduction of pro-inflammatory cytokines is somewhat similar to, though in a mild form, health conditions in obesity and related metabolic disorders like type-2 diabetes, which are also considered important risk factors for severe illness in COVID-19. Interestingly, neutrophils perhaps play a significant role in this pathogenesis. On the other hand, it is thought that COVID-19-related critical illness is associated with pathological hyperactivity of the complement system and coagulopathy. Although the precise molecular interactions between the complement and coagulation systems are not clear, we observe an intimate cross-talk between these two systems in critically ill COVID-19 patients. It is believed that both of these biological systems are connected with the cytokine storm in severe COVID-19 disease and actively participate in this vicious cycle. In order to hinder the pathological progression of COVID-19, a number of anticoagulation agents and complement inhibitors have been used with varying success. Among these drugs, low molecular weight heparin enoxaparin, factor Xa inhibitor apixaban, and complement C5 inhibitor eculizumab have been commonly used in patients with COVID-19. Our overall experience might help us in the future to tackle any such conditions.

Urinary complement proteins in IgA nephropathy progression from a relative quantitative proteomic analysis.

Aim: IgA nephropathy (IgAN) is one of the leading causes of end-stage renal disease (ESRD). Urine testing is a non-invasive way to track the biomarkers used for measuring renal injury. This study aimed to analyse urinary complement proteins during IgAN progression using quantitative proteomics. Methods: In the discovery phase, we analysed 22 IgAN patients who were divided into three groups (IgAN 1-3) according to their estimated glomerular filtration rate (eGFR). Eight patients with primary membranous nephropathy (pMN) were used as controls. Isobaric tags for relative and absolute quantitation (iTRAQ) labelling, coupled with liquid chromatography-tandem mass spectrometry, was used to analyse global urinary protein expression. In the validation phase, western blotting and parallel reaction monitoring (PRM) were used to verify the iTRAQ results in an independent cohort (N = 64). Results: In the discovery phase, 747 proteins were identified in the urine of IgAN and pMN patients. There were different urine protein profiles in IgAN and pMN patients, and the bioinformatics analysis revealed that the complement and coagulation pathways were most activated. We identified a total of 27 urinary complement proteins related to IgAN. The relative abundance of C3, the membrane attack complex (MAC), the complement regulatory proteins of the alternative pathway (AP), and MBL (mannose-binding lectin) and MASP1 (MBL associated serine protease 2) in the lectin pathway (LP) increased during IgAN progression. This was especially true for MAC, which was found to be involved prominently in disease progression. Alpha-N-acetylglucosaminidase (NAGLU) and α-galactosidase A (GLA) were validated by western blot and the results were consistent with the iTRAQ results. Ten proteins were validated in a PRM analysis, and these results were also consistent with the iTRAQ results. Complement factor B (CFB) and complement component C8 alpha chain (C8A) both increased with the progression of IgAN. The combination of CFB and mucosal addressin cell adhesion molecule-1 (MAdCAM-1) also showed potential as a urinary biomarker for monitoring IgAN development. Conclusion: There were abundant complement components in the urine of IgAN patients, indicating that the activation of AP and LP is involved in IgAN progression. Urinary complement proteins may be used as biomarkers for evaluating IgAN progression in the future.

Risk and protection of different rare protein-coding variants of complement component C4A in age-related macular degeneration.

Introduction: Age-related macular degeneration (AMD) is the leading cause of central vision loss in the elderly. One-third of the genetic contribution to this disease remains unexplained. Methods: We analyzed targeted sequencing data from two independent cohorts (4,245 cases, 1,668 controls) which included genomic regions of known AMD loci in 49 genes. Results: At a false discovery rate of <0.01, we identified 11 low-frequency AMD variants (minor allele frequency <0.05). Two of those variants were present in the complement C4A gene, including the replacement of the residues that contribute to the Rodgers-1/Chido-1 blood group antigens: [VDLL1207-1210ADLR (V1207A)] with discovery odds ratio (OR) = 1.7 (p = 3.2 × 10-5) which was replicated in the UK Biobank dataset (3,294 cases, 200,086 controls, OR = 1.52, p = 0.037). A novel variant associated with reduced risk for AMD in our discovery cohort was P1120T, one of the four C4A-isotypic residues. Gene-based tests yielded aggregate effects of nonsynonymous variants in 10 genes including C4A, which were associated with increased risk of AMD. In human eye tissues, immunostaining demonstrated C4A protein accumulation in and around endothelial cells of retinal and choroidal vasculature, and total C4 in soft drusen. Conclusion: Our results indicate that C4A protein in the complement activation pathways may play a role in the pathogenesis of AMD.

Efficacy of a vegetal mixture composed of Zingiber officinale, Echinacea purpurea, and Centella asiatica in a mouse model of neuroinflammation: In vivo and ex vivo analysis.

Experimental evidence suggests that neuroinflammation is a key pathological event of many diseases affecting the nervous system. It has been well recognized that these devastating illnesses (e.g., Alzheimer's, Parkinson's, depression, and chronic pain) are multifactorial, involving many pathogenic mechanisms, reason why pharmacological treatments are unsatisfactory. The purpose of this study was to evaluate the efficacy of a vegetal mixture capable of offering a multiple approach required to manage the multifactoriality of neuroinflammation. A mixture composed of Zingiber officinale (150 mg kg-1), Echinacea purpurea (20 mg kg-1), and Centella asiatica (200 mg kg-1) was tested in a mouse model of systemic neuroinflammation induced by lipopolysaccharide (LPS, 1 mg kg-1). Repeated treatment with the vegetal mixture was able to completely counteract thermal and mechanical allodynia as reported by the Cold plate and von Frey tests, respectively, and to reduce the motor impairments as demonstrated by the Rota rod test. Moreover, the mixture was capable of neutralizing the memory loss in the Passive avoidance test and reducing depressive-like behavior in the Porsolt test, while no efficacy was shown in decreasing anhedonia as demonstrated by the Sucrose preference test. Finally, LPS stimulation caused a significant increase in the activation of glial cells, of the central complement proteins and of inflammatory cytokines in selected regions of the central nervous system (CNS), which were rebalanced in animals treated with the vegetal mixture. In conclusion, the vegetal mixture tested thwarted the plethora of symptoms evoked by LPS, thus being a potential candidate for future investigations in the context of neuroinflammation.

The effects of exercise on complement system proteins in humans: a systematic scoping review.

BACKGROUND: The complement system is comprised of the classical, lectin and alternative pathways that result in the formation of: pro-inflammatory anaphylatoxins; opsonins that label cells for phagocytic removal; and, a membrane attack complex that directly lyses target cells. Complement-dependent cytotoxicity (CDC) - cell lysis triggered by complement protein C1q binding to the Fc region of antibodies bound to target cells - is another effector function of complement and a key mechanism-of-action of several monoclonal antibody therapies. At present, it is not well established how exercise affects complement system proteins in humans. METHODS: A systematic search was conducted to identify studies that included original data and investigated the association between soluble complement proteins in the blood of healthy humans, and: 1) an acute bout of exercise; 2) exercise training interventions; or, 3) measurements of habitual physical activity and fitness. RESULTS: 77 studies were eligible for inclusion in this review, which included a total of 10,236 participants, and 40 complement proteins and constituent fragments. Higher levels of exercise training and cardiorespiratory fitness were commonly associated with reduced C3 in blood. Additionally, muscle strength was negatively associated with C1q. Elevated C3a-des-Arg, C4a-des-Arg and C5a, lower C1-inhibitor, and unchanged C3 and C4 were reported immediately post-laboratory based exercise, compared to baseline. Whereas, ultra-endurance running and resistance training increased markers of the alternative (factor B and H), classical (C1s), and leptin (mannose binding lectin) pathways, as well as C3 and C6 family proteins, up to 72-h following exercise. Heterogeneity among studies may be due to discrepancies in blood sampling/handling procedures, analytical techniques, exercise interventions/measurements and fitness of included populations. CONCLUSIONS: Increased anaphylatoxins were observed immediately following an acute bout of exercise in a laboratory setting, whereas field-based exercise interventions of a longer duration (e.g. ultra-endurance running) or designed to elicit muscle damage (e.g. resistance training) increased complement proteins for up to 72-h. C3 in blood was mostly reduced by exercise training and associated with increased cardiorespiratory fitness, whereas C1q appeared to be negatively associated to muscle strength. Thus, both acute bouts of exercise and exercise training appear to modulate complement system proteins. Future research is needed to assess the clinical implications of these changes, for example on the efficacy of monoclonal antibody therapies dependent on CDC.

Plasma Small Extracellular Vesicles with Complement Alterations in GRN/C9orf72 and Sporadic Frontotemporal Lobar Degeneration.

Cutting-edge research suggests endosomal/immune dysregulation in GRN/C9orf72-associated frontotemporal lobar degeneration (FTLD). In this retrospective study, we investigated plasma small extracellular vesicles (sEVs) and complement proteins in 172 subjects (40 Sporadic FTLD, 40 Intermediate/Pathological C9orf72 expansion carriers, and 49 Heterozygous/Homozygous GRN mutation carriers, 43 controls). Plasma sEVs (concentration, size) were analyzed by nanoparticle tracking analysis; plasma and sEVs C1q, C4, C3 proteins were quantified by multiplex assay. We demonstrated that genetic/sporadic FTLD share lower sEV concentrations and higher sEV sizes. The diagnostic performance of the two most predictive variables (sEV concentration/size ratio) was high (AUC = 0.91, sensitivity 85.3%, specificity 81.4%). C1q, C4, and C3 cargo per sEV is increased in genetic and sporadic FTLD. C4 (cargo per sEV, total sEV concentration) is increased in Sporadic FTLD and reduced in GRN+ Homozygous, suggesting its specific unbalance compared with Heterozygous cases. C3 plasma level was increased in genetic vs. sporadic FTLD. Looking at complement protein compartmentalization, in control subjects, the C3 and C4 sEV concentrations were roughly half that in respect to those measured in plasma; interestingly, this compartmentalization was altered in different ways in patients. These results suggest sEVs and complement proteins as potential therapeutic targets to mitigate neurodegeneration in FTLD.

Methods for Assessment of Interactions of Proteins with Heme: Application for Complement Proteins and Immunoglobulins.

Heme (Fe protoporphyrin IX) serves as a prosthetic group of numerous proteins implicated in oxidative metabolism. This molecule is abundantly present in the red blood cells where it serves as a cofactor of hemoglobin. As consequence of various pathological conditions, the membrane of red blood cells can be damaged and therefore large quantities of hemoglobin and subsequently heme released in the extracellular space. Since heme is a highly reactive compound, when released extracelluarly it can influence the functional activity of different plasma components. Thus, previous investigations have demonstrated that heme can interact with components of complement system and immunoglobulins, profoundly affecting their functions. Here we propose two basic protocols that can be used for characterization of interaction of free heme with complement proteins and immunoglobulins. The first technique is based on UV-Vis absorbance spectroscopy. It allows general characterization of the heme binding to the protein and estimation of the number of heme binding sites. The second protocol consists in the use of biosensor assay based on surface plasmon resonance. This protocol would be useful for evaluation of heme binding kinetics and equilibrium affinity. Besides for complement components and immunoglobulins, the presented protocols can be utilized for characterization of the interaction of heme with other proteins.