Complement Protein C3 Research Articles

A novel fasting regimen revealed protein reservation and complement C3 down-regulation after 14-day's continual dietary deprivation.

The aim is to evaluate the effect of a novel 14-day fasting regimen on the balance between skeletal muscle and adipose tissue composition which might associate with inflammatory factors. Our analysis includes basic physical examinations, clinical laboratory analysis, bioelectrical impedance and biochemical analytic assessments of healthy volunteers. Eight healthy subjects were randomly selected from a pool of volunteers to undergo a continual dietary deprivation (CDD) regimen. Individuals were assigned to take Flexible Abrosia (FA, prebiotic combination) plus appropriate mineral supplement of potassium and magnesium at 3 mealtime every day to prevent potential injury from starved intestinal flora and avoid spasms of smooth muscle due to hunger. Physical and medical examinations were conducted and blood samples were collected at following timepoints: before CDD as self-control (0D), day 7 and day 14 during fasting, and 7-21days and/or 2~3mo after refeeding. The combination of FA and mineral supplements significantly decreased self-reported physical response of starvation, with tolerable hunger-mediated sensations experienced during CDD. Bioelectrical and biochemical results indicated significant reduction in both muscle lean and fat mass on day 7. Meanwhile, markers related to fat composition consistently decreased during and after CDD. In addition, most biochemical marker levels, including serum proteins, reached their inflection points at the 7th day of CDD as compared to the control measurements. Levels of these factors started to show a relative plateau, or reversed direction upon the 14th day of CDD. The exceptions of above factors were myostatin and complement protein C3, which remained at lower concentrations in the blood throughout CDD, and were unable to fully recover toward baseline levels even after 3 months' refeeding. Our results indicated that human subjects undergoing prolonged dietary restriction were well protected by FA and mineral ions from gut injury or physical discomfort of starvation. Most factors showed a relative plateau response at the end of 14D-CDD. The muscle tissues were well preserved during prolonged fasting, and an improved protein/lipid ratio was observed. Upon refeeding, constant lower levels of myostatin and complement C3 were maintained after CDD implies a long-term beneficial effect in dealing with anti-aging and inflammation.

Plasma complement C3 and C3a are increased in major depressive disorder independent of childhood trauma

BackgroundDysregulated complement system is linked to pathophysiology of major depressive disorder (MDD). Childhood trauma has been associated with an increased incidence of adult depression via a putative mechanism of immune activation. This study aimed to measure and compare peripheral levels of complement C3, C3a, C1q and C-reactive protein (CRP) in MDD patients and healthy controls and explore the relationship between these molecule levels and childhood trauma history in the participants.MethodsThe participants were 49 medication-free MDD patients and 45 healthy controls. All participants were asked to finish the Childhood Trauma Questionnaire, followed by blood sampling for measurement of plasma complement C3, C3a, C1q and CRP by means of enzyme-linked immunosorbent assay.ResultsPeripheral plasma concentration of C3 and C3a in medication-free MDD group was significantly higher than that in the healthy controls; whereas the concentration of plasma C1q and CRP in depressed patients was comparable to that in healthy controls. All these inflammatory factors were not associated to childhood trauma experience in patients with MDD.ConclusionOur data suggest that complement C3 and C3a may be implicated in the pathophysiology of MDD, although traumatic childhood experiences were not associated with the circulating levels of complement C3, C3a, C1q and CRP.

Interactions of Candida tropicalis pH-related antigen 1 with complement proteins C3, C3b, factor-H, C4BP and complement evasion

Candida, as a part of the human microbiota, can cause opportunistic infections that are either localised or systemic candidiasis. Emerging resistance to the standard antifungal drugs is associated with increased mortality rate due to invasive Candida infections, particularly in immunocompromised patients. While there are several species of Candida, an increasing number of Candida tropicalis isolates have been recently reported from patients with invasive candidiasis or inflammatory bowel diseases. In order to establish infections, C. tropicalis has to adopt several strategies to escape the host immune attack. Understanding the immune evasion strategies is of great importance as these can be exploited as novel therapeutic targets. C. albicans pH-related antigen 1 (CaPra1), a surface bound and secretory protein, has been found to interact strongly with the immune system and help in complement evasion. However, the role of C. tropicalis Pra1 (CtPra1) and its interaction with the complement is not studied yet. Thus, we characterised how pH-related antigen 1 of C. tropicalis (CtPra1) interacts with some of the key complement proteins of the innate immune system. CtPra1 was recombinantly produced using a Kluyveromyces lactis yeast expression system. Recombinant CtPra1, was found to bind human C3 and C3b, central molecules of the complement pathways that are important components of the innate immune system. It was also found to bind human complement regulatory proteins factor-H and C4b-binding protein (C4BP). CtPra1-factor-H and CtPra1-C4BP interactions were found to be ionic in nature as the binding intensity affected by high sodium chloride concentrations. CtPra1 inhibited functional complement activation with different effects on classical (∼20 %), lectin (∼25 %) and alternative (∼30 %) pathways. qPCR experiments using C. tropicalis clinical isolates (oral, blood and peritoneal fluid) revealed relatively higher levels of expression of CtPra1 gene when compared to the reference strain. Native CtPra1 was found to be expressed both as membrane-bound and secretory forms in the clinical isolates. Thus, C. tropicalis appears to be a master of immune evasion by using Pra1 protein. Further investigation using in-vivo models will help ascertain if these proteins can be novel therapeutic targets.

COVID-19 Associated Atypical Hemolytic Uremic Syndrome

Introduction: Atypical hemolytic uremic syndrome (atypical HUS) is a complement-mediated thrombotic microangiopathy (TMA) that affects multiple organs including the kidneys. We present a case of 37-year-old male with recent COVID-19 infection who presented with epistaxis. His blood work was consistent with TMA and acute kidney injury (AKI). Hemodialysis and plasmapheresis (PLEX) were initiated for presumed thrombotic thrombocytopenic purpura (TTP) but after excluding ADAMTS-13 deficiency, eculizumab was started for possible atypical HUS. Renal biopsy and genetic testing ultimately confirmed diagnosis of atypical HUS. Case presentation: A 37-year-old male with past medical history of recent COVID infection presented to the emergency room with complaint of epistaxis. Physical exam showed mild epistaxis from right naris and bilateral leg swelling. Initial laboratory findings were hemoglobin (Hb) 5 g/dL, platelets 70 B/L, Lactate dehydrogenase (LDH) 581 IU/L, haptoglobin < 10 mg/dL, creatinine (Cr) 36.80 mg/dL, blood urea nitrogen (BUN) 232 mg/dL, potassium (K) 6.6 mmol/L, calcium (Ca) 6 mg/dL and normal prothrombin time (PT) and partial thromboplastin time (PTT). Complement C3 levels were low but normal C4 levels. Peripheral blood smear showed schistocytes confirming TMA. He was started on both PLEX and hemodialysis (HD) along with prednisone 80 mg daily. PLEX was stopped on day 4 after normal ADAMTS-13 levels resulted, helping rule out TTP. Based on history and laboratory findings, diagnosis of atypical HUS was favored and eculizumab was started. He received meningococcal vaccine and prophylactic penicillin while on eculizumab. Hemodialysis was continued with eventual improvement of his labs to Hb 7, platelets 119, LDH 339, haptoglobin < 10, Cr 9.07, BUN 85, K 5.8, and Ca 8.0. He was discharged on bimonthly dose of eculizumab and hemodialysis three times a week. Two months later, his labs were Hb 10.4, platelets 184, LDH 490, haptoglobin < 10, Cr 11.60, BUN 82, K 5.2, and Ca 9.2. PT and PTT remained normal. Given persistence of laboratory findings, renal biopsy and genetic testing for complement proteins was done. Kidney biopsy showed diffuse global renal microangiopathy with negative C3. Genetic testing showed a heterozygous C3 variant, homozygous deletion of CFHR1-CFHR4, negative factor H autoantibodies (FHAA), and single nucleotide variant in C5 gene. Discussion: Atypical HUS is a disease characterized by the triad of microangiopathic hemolytic anemia, thrombocytopenia, and AKI. The complement system is a part of the immune system that enhances the ability of phagocytic cells to clear microbes and damaged cells. Atypical HUS is caused by dysregulation of the alternative complement pathway that leads to microvascular thrombosis. Various mutations in complement and its regulatory proteins have been discovered in atypical HUS patients. Heterozygous C3 variants result in persistently low C3 levels and cause severe disease. Most individuals with a homozygous deletion of CFHR1 and/or CFHR3, CFHR4 genes have a pathogenic role in the development of the anti-factor H autoantibodies. These autoantibodies interfere with the binding of CFH to the C3 convertase and are associated with defective CFH-dependent cell protection. Some case reports of COVID-19 patients showed that complement activation was significantly stronger in lung alveolar septa, hyaline membranes, and in alveolar lumina via the lectin and alternative complement pathways. Treatment of atypical HUS is symptomatic but in severe disease eculizumab or ravulizumab have roles by binding to the complement protein C5, inhibiting its cleavage to C5a and C5b, and preventing formation of terminal complement complex C5b-9. Patients on these therapies are at risk of meningococcal disease, thus it is recommended to get meningococcal vaccines and prophylactic penicillin for the duration of therapy. Our patient had homozygous deletion of CFHR1-CFHR4 genes with no factor H autoantibodies. However, he did have C3 variant with gain of function mutation which caused overt activation of alternative pathway as indicated by his low C3 levels with normal C4 levels. He responded partially to eculizumab and discussion on possible kidney transplant has been started.

Characterization for the Similarity Assessment between SB12 (Eculizumab Biosimilar) and Eculizumab Using State-of-the-Art Analytical Method

Background: SB12 is being developed as a proposed biosimilar to eculizumab reference product (RP), a humanized monoclonal antibody (IgG2/4 kappa immunoglobulin) that binds to the human C5 complement protein. Binding to this protein inhibits complement-mediated intravascular hemolysis by blocking its cleavage into C5a and C5b. Eculizumab RP is indicated for the treatment of patients with paroxysmal nocturnal hemoglobinuria (PNH) to reduce hemolysis, atypical hemolytic uremic syndrome (aHUS) to inhibit complement-mediated thrombotic microangiopathy, generalized Myasthenia Gravis (gMG) who are anti-acetylcholine receptor (AChR) antibody positive, and Neuromyelitis Optica Spectrum Disorder (NMOSD) in adult patients who are anti-aquaporin-4 (AQP4) positive antibody. Objective: This study was to demonstrate structural, physicochemical, and functional similarity between SB12 and eculizumab RP using state-of-the-art analytical procedures. Methods: Comprehensive analytical characterization was conducted with side-by-side comparison with SB12, EU and US eculizumab RP using various analytical methods (more than 40 state-of-the-art assays). Comparisons included purity, product-related impurity, charge heterogeneity, primary structure, post-translational modification, higher order structure, quantity, Fab-related biological activities (potency and binding activity), and Fc-related biological activities. Results: Based on the analytical similarity assessment, the structural, physicochemical, and biological characterization results demonstrated that SB12 is highly similar to EU and US eculizumab RP by showing qualities that met the criteria for similarity assessment set with the eculizumab RP. In the structural aspects, it was confirmed that there is no difference between post-translational modification profiles and higher order structures of SB12 compared to the reference product. Product-related size, charge variants, and aggregates were also confirmed to be similar. In case of MoA-related biological activities showed that SB12 is highly similar to EU and US eculizumab RP with respect to overall critical and non-critical quality attributes performed. Moreover, similarity of comparative binding tendency of SB12 and eculizumab RP to Fc gamma receptors and C1q was confirmed through additional characterization. Based on these results, SB12 is expected to have highly similar safety and efficacy comparable to eculizumab RP. Conclusion: In summary, the overall characterization results show that SB12 is highly similar to EU and US eculizumab RP in terms of structural, physicochemical, biophysical, and biological attributes

Probing human proteins for short encrypted antimicrobial peptides reveals Hs10, a peptide with selective activity for gram-negative bacteria

BackgroundSome cationic and amphiphilic α-helical segments of proteins adsorb to prokaryotic membranes when synthesized as individual polypeptide sequences, resulting in broad and potent antimicrobial activity. However, amphiphilicity, a determinant physicochemical property for peptide-membrane interactions, can also be observed in some β-sheets. MethodsThe software Kamal was used to scan the human reference proteome for short (7–11 amino acid residues) cationic and amphiphilic protein segments with the characteristic periodicity of β-sheets. Some of the uncovered peptides were chemically synthesized, and antimicrobial assays were conducted. Biophysical techniques were used to probe the molecular interaction of one peptide with phospholipid vesicles, lipopolysaccharides (LPS) and the bacterium Escherichia coli. ResultsThousands of compatible segments were found in human proteins, five were synthesized, and three presented antimicrobial activity in the micromolar range. Hs10, a nonapeptide fragment of the Complement C3 protein, could inhibit only the growth of tested Gram-negative microorganisms, presenting also little cytotoxicity to human fibroblasts. Hs10 interacted with LPS while transitioning from an unstructured segment to a β-sheet and increased the hydrodynamic radius of LPS particles. This peptide also promoted morphological alterations in E. coli cells. Conclusions: Data presented herein introduce yet another molecular template to probe proteins in search for encrypted membrane-active segments and demonstrates that, using this approach, short peptides with low cytotoxicity and high selectivity to prokaryotic cells might be obtained. General SignificanceThis work widens the biotechnological potential of the human proteome as a source of antimicrobial peptides with application in human health.

NRSF regulates age-dependently cognitive ability and its conditional knockout in APP/PS1 mice moderately alters AD-like pathology.

NRSF/REST (neuron-restrictive silencer element, also known as repressor element 1-silencing transcription factor), plays a key role in neuronal homeostasis as a transcriptional repressor of neuronal genes. NRSF/REST relates to cognitive preservation and longevity of humans, but its specific functions in age-dependent and Alzheimer's disease (AD)-related memory deficits remain unclear. Here, we show that conditional NRSF/REST knockout either in the dorsal telencephalon or specially in neurons induced an age-dependently diminished retrieval performance in spatial or fear conditioning memory tasks and altered hippocampal synaptic transmission and activity-dependent synaptic plasticity. The NRSF/REST deficient mice were also characterized by an increase of activated glial cells, complement C3 protein and the transcription factor C/EBPβ in the cortex and hippocampus. Reduction of NRSF/REST by conditional depletion upregulated the activation of astrocytes in APP/PS1 mice, and increased the C3-positive glial cells, but did not alter the Aβ loads and memory retrieval performances of 6- and 12-month-old APP/PS1 mice. Simultaneously, overexpression of NRSF/REST improved cognitive abilities of aged wild type, but not in AD mice. These findings demonstrated that NRSF/REST is essential for the preservation of memory performance and activity-dependent synaptic plasticity during aging and takes potential roles in the onset of age-related memory impairments. However, while altering the glial activation, NRSF/REST deficiency does not interfere with the Aβ deposits and the electrophysiological and cognitive AD-like pathologies.

Immunological profile of erythema nodosum and their relationship of C-reactive protein level and erythrocyte sedimentation rate: A retrospective analysis of 61 patients.

This experiment was carried out to investigate changes in lymphocyte subpopulation, immunoglobulins (Igs), and complements, and also to explore the relationship between these immune indices and C-reactive protein and erythrocyte sedimentation rate in 61 patients with erythema nodosum. For this aim, a 4-year, retrospective study contained 61 patients with erythema nodosum, and 61 healthy control subjects were included from the out-patient clinic. The subpopulation of the T, B and natural killer lymphocytes and levels of IgA, IgG, IgM, complement C3, complement C4, C-reactive protein, and erythrocyte sedimentation rate from peripheral blood of them were detected. A correlation analysis was done between lymphocyte subpopulation, levels of IgA, IgG, and IgM, complement C3, complement C4 and C-reactive protein level and erythrocyte sedimentation rate in the patient group. Results showed that the percentage of CD4+ cells, CD4+/CD8+ ratio, the level of C-reactive protein and the erythrocyte sedimentation rate in the patients were higher than in controls (P<0.05). While the percentage of CD8+ cells and the serum levels of complement C3 were lower than in controls (P<0.05). There were no differences in the percentages of CD3+, B and natural killer cells and the serum levels of IgA, IgG and IgM, and complement C4 between the patients and the controls (P>0.05). IgM level was positively correlated with C-reactive protein (P<0.05) but did not correlate with an erythrocyte sedimentation rate (P>0.05). There was no correlation between lymphocyte subpopulation, levels of IgA, IgG, complement C3, complement C4 and C-reactive protein level and erythrocyte sedimentation rate (P>0.05). In conclusion, there was dysregulation of both cellular immunity and humoral immunity in patients with erythema nodosum. IgM level has a positive correlation with C-reactive protein.

Proteomics analysis revealed the presence of inflammatory and oxidative stress markers in the plasma of migraine patients during the pain period

BackgroundThere is increasing evidence that some biomarkers are implicated in migraine pathogenesis. This study looks at plasma proteome in migraine patients for potential protein biomarkers. MethodsThis case-control study has two phases. In phase I, plasma samples were collected from three groups, including twenty-three episodic migraineurs, thirty-five chronic migraineurs, and twenty-nine healthy subjects. In phase II, plasma samples were prepared from two groups, including five episodic and five chronic migraine cases, during the pain and 24 h after the pain-free periods. Two-dimensional gel electrophoresis (2-DE) was performed on plasma proteins. The possible corresponding proteins for the differentially expressed spots between groups investigated by the Melanie software were predicted by 2-DE gels of the EXPASY database. LC-MS/MS additionally analyzed phase II data. ResultsExpression levels of haptoglobin, clusterin, fibrinogen alpha chain, fibrinogen beta chain, complement c3, transthyretin, α1-microglobulin, and retinol-binding protein 4 were shown considerable changes in migraine patients compared to controls or their pain-free period. ConclusionDifferences in expression levels for several proteins were observed across groups. Most of these are associated with inflammation, oxidative stress, and neuroprotection, which can be considered potential disease biomarkers. However, further research is necessary for this respect.

Analysis of the relationship between human complement components C3 &amp; C4 deficiency and C– reactive protein levels as an indicator of the increase of microbial infections

The complement system is an important component of the immune system that consists of a group of plasma proteins produced by the liver and serving a defense function. As a result, the deficiency in any of the complement system components leads to the loss of the function of the deficient protein causing a specific clinical problem or disease. For that reason, this study was designed to investigate C3 and C4 deficiencies in young patients and to inspect the possibility of using C-Reactive Protein (CRP) test and the variation of C3 and C4 concentrations in children`s blood as an indicator of the onset of infection. This study was conducted in the children hospital in Benghazi city, Libya. The blood samples were collected from 446 children in all hospital units to examine the proportion of complement deficiency among those children and C3, C4 deficiency was set as an indicator of infection occurrence. The results of this study have shown a deficiency in complement proteins C3 and C4 were in 44 (10%) samples and 2 (0.4%) samples respectively, the female candidates had a higher rate of C3 and C4 deficiency than males. In addition, the CRP levels were elevated in 167 (55.9%) cases. Furthermore, the association between the increase of CRP levels and low levels of C3 and C4 was observed in 26 (8.7%) cases and 2 (0.6%) cases. These results suggest a strong association between complement proteins C3 and C4 deficiency and an elevation of CRP levels (P-Value=0.00), which supports the concept of using such association as an indicator of microbial infection

Charting the Proteoform Landscape of Serum Proteins in Individual Donors by High-Resolution Native Mass Spectrometry.

Most proteins in serum are glycosylated, with several annotated as biomarkers and thus diagnostically important and of interest for their role in disease. Most methods for analyzing serum glycoproteins employ either glycan release or glycopeptide centric mass spectrometry-based approaches, which provide excellent tools for analyzing known glycans but neglect previously undefined or unknown glycosylation and/or other co-occurring modifications. High-resolution native mass spectrometry is a relatively new technique for the analysis of intact glycoproteins, providing a “what you see is what you get” mass profile of a protein, allowing the qualitative and quantitative observation of all modifications present. So far, a disadvantage of this approach has been that it centers mostly on just one specific serum glycoprotein at the time. To address this issue, we introduce an ion-exchange chromatography-based fractionation method capable of isolating and analyzing, in parallel, over 20 serum (glyco)proteins, covering a mass range between 30 and 190 kDa, from 150 μL of serum. Although generating data in parallel for all these 20 proteins, we focus the discussion on the very complex proteoform profiles of four selected proteins, i.e., α-1-antitrypsin, ceruloplasmin, hemopexin, and complement protein C3. Our analyses provide an insight into the extensive proteoform landscape of serum proteins in individual donors, caused by the occurrence of various N- and O-glycans, protein cysteinylation, and co-occurring genetic variants. Moreover, native mass intact mass profiling also provided an edge over alternative approaches revealing the presence of apo- and holo-forms of ceruloplasmin and the endogenous proteolytic processing in plasma of among others complement protein C3. We also applied our approach to a small cohort of serum samples from healthy and diseased individuals. In these, we qualitatively and quantitatively monitored the changes in proteoform profiles of ceruloplasmin and revealed a substantial increase in fucosylation and glycan occupancy in patients with late-stage hepatocellular carcinoma and pancreatic cancer as compared to healthy donor samples.

Erythrocyte complement receptor 1 (ECR1) and erythrocyte-bound C4d (EC4d) in the prediction of poor pregnancy outcomes in systemic lupus erythematosus (SLE)

BackgroundComplement activation has been associated with adverse pregnancy outcomes (APO) in SLE. Pregnant women with SLE were studied to evaluate whether complement dysregulation within the first two pregnancy trimesters predicts...

Inactivation of the Complement Lectin Pathway by Candida tropicalis Secreted Aspartyl Protease-1

Candida tropicalisis an opportunistic fungal pathogen and is one of the most frequently isolated non-albicans species. It can cause localised as well as invasive systemic infections particularly in immunocompromised patients. Increased resistance to common anti-fungal drugs is an emerging problem. In order to establish disseminated infections, Candida has evolved several strategies to escape the host immune system. A detailed understanding of how C. tropicalis escapes the host immune attack is needed as it can help develop novel anti-fungal therapies. Secreted aspartyl proteinases (Saps) of C. albicans have been shown to be determinants of virulence and immune evasion. However, the immune evasion properties of C. tropicalis Saps have been poorly characterised. This study investigated the immune evasion properties of C. tropicalis secreted aspartic protease 1 (Sapt1).Sapt1 was recombinantly produced using a Kluyveromyces lactis yeast expression system. A range of complement proteins and immunogloublins were screened to test if Sapt1 had any proteolytic activity. Sapt1 efficiently cleaved human mannose-binding lectin (MBL) and collectin-11, which are the initiating molecules of the lectin pathway of the complement system, but not l-ficolin. In addition, Sapt1 cleaved DC-SIGN, the receptor on antigen presenting dendritic cells. Proteolysis was prominent in acidic condition (pH 5.2), a characteristic of aspartyl protease. No proteolytic activity was detected against complement proteins C1q, C3, C3b, IgG and IgA. In view of the ability of Sapt1 to cleave MBL and collectin-11, we found that Sapt1 could prevent activation of the complement lectin pathway. RT-qPCR analysis using three different C. tropicalis clinical isolates (oral, blood and peritoneal dialysis fluid) revealed relatively higher levels of mRNA expression of Sapt1 gene when compared to a reference strain; Sapt1 protein was found to be secreted by all the tested strains. Lectin pathway and its initiating components are crucial to provide front line defence against Candida infections. For the first time, we have shown that a Candida protease can proteolytically degrade the key initiating components of lectin pathway and inhibit complement activation. Findings from this study highlight the importance of exploring Sapt1 as a potential therapeutic target. We conclude that C. tropicalis secretes Sapt1 to target the complement lectin pathway, a key pattern recognition and clearance mechanism, for its survival and pathogenesis.

The Role of Complement Split-products as Biomarkers for Acute Antibody-mediated Rejection of Kidney Allografts.

Background.Acute antibody-mediated rejection (AMR) is mediated by the activation of the classical complement system in addition to noncomplement-dependent inflammatory pathways. Complement fixation by donor-specific antibodies leads to cleavage of the complement proteins C4, C3, and C5 to produce multiple complement split-products (CSP) and the end-effector membrane attack complex, C5b-9. In this study, we investigate CSP as potential biomarkers for AMR.Methods.In an Institutional Review Board–approved, prospective, controlled study, CSP levels were measured in blood and urine samples from consecutive kidney transplant recipients with biopsy-proven AMR (n = 10), acute cellular rejection (ACR) (n = 5), or no rejection (n = 5). After obtaining informed consent, samples were collected at the time of biopsy (day 0) and days 15 (end of rejection treatment) and 30 postbiopsy for AMR and ACR patients. ELISA was used to measure C5a, C4d, and soluble C5b-9 concentrations in blood and urine, in addition to factor Bb (Bb) concentration in blood only. Kidney transplant histopathology was evaluated using the Banff 2013 classification. Rejection treatment and follow-up were performed per standard of care.Results.Blood and urine CSP levels adjusted to urine creatinine were not elevated in AMR compared to no rejection and ACR arms. There was significant variability in CSP concentration within each of the study groups.Conclusion.Our study does not support the utility of CSP as surrogate biomarkers of AMR; however, it is limited by the small sample size and larger studies may be warranted.

Associations of HDL subclasses and lipid content with complement proteins over the menopause transition: The SWAN HDL ancillary study: HDL and complement proteins in women

The menopause transition (MT) could trigger low-grade chronic inflammation which may modify high-density lipoproteins (HDL) and lead to additional inflammatory responses contributing to atherosclerosis development. To test whether complement proteins C3 and C4 increase around the final menstrual period (FMP), and whether changes in HDL subclasses and lipid content associate with C3 and C4 levels over time in midlife women. The study included 471 women (at baseline: age 50.2(2.7) years; 87.3% pre or peri-menopausal) who had nuclear magnetic resonance spectroscopy HDL subclasses, lipid content, and C3 and C4 measured up to 5 times over the MT. Adjusted annual changes in C3 and C4 varied by time segments relative to FMP with significant increases, steeper for C3, only observed within 1 year before to 2 years after the FMP. Greater decreases in large HDL particles (HDL-P), HDL size, and HDL-phospholipids, and greater increases in small HDL-P and HDL-Triglycerides were associated with higher C3 and C4 over time, although associations with C4 were weaker than those with C3. Complement proteins C3 and C4 significantly rise around menopause with C3 showing the steepest rise. Changes in HDL subclasses, overall size, and lipid content, over the MT may play a role in modulating inflammation responses known to be related to atherosclerosis. These results raise the possibility that novel therapeutic agents focusing on HDL might contribute to CVD protection by modulating inflammation.

Complement factor B inhibitor LNP023 improves lupus nephritis in MRL/lpr mice

BackgroundLupus nephritis (LN) is the most common complication of systemic lupus erythematosus (SLE), and the abnormal activation of the alternative complement pathway is associated with the pathogenesis of LN. As an inhibitor of complement factor B (CFB) in the alternative pathway, LNP023 has been used in the treatment of a variety of renal diseases with abnormal complement system involvement, such as paroxysmal nocturnal hemoglobinuria, IgA nephropathy, and membranous nephropathy. The aim of our study was to explore whether LNP023 improved LN in MRL/lpr mice by inhibiting the activation of the alternative complement pathway. MethodsThe mice were divided into a normal control group (Normal group) (n = 6), MRL/lpr model group (n = 6), and LNP023 group (n = 6). The LNP023 group was administered LNP023 for 2 weeks by gavage; the MRL/lpr model group was administered saline for 2 weeks by gavage; and the Normal group was administered saline for 2 weeks by gavage. External signs, renal pathology, renal function, renal immune complex and complement deposition, serum anti-dsDNA, serum ANA concentration, and the expression of core complement factors in the alternative complement pathway were analyzed in the 3 groups of animals. The core targets of LNP023 in the treatment of LN were screened using network pharmacology. The pathogenicity of the core targets in LN was verified by analyzing the mRNA expression of the core targets in the peripheral blood mononuclear cells (PBMCs) of normal individuals, SLE patients, and LN patients. The mRNA and protein expression of core targets in the Normal group, MRL/lpr group, and LNP023 group were analyzed to verify whether LNP023 exerted it LN therapeutic effect through the regulation of core targets. ResultsCompared with the MRL/lpr group, the LNP023 group had reduced lupus-like signs, improved renal function, decreased serum anti-dsDNA and ANA concentrations, and reduced renal IgM, IgG, IgG1, C1q, C3, and C4 deposition. Renal pathology showed that LNP023 attenuated pathological damage in the kidneys of MRL/lpr mice. Compared with the MRL/lpr model group, the treatment group had no crescent formation, less immune deposition, no nuclear fragmentation, and less inflammatory cell infiltration. The expression of complement proteins C3, C3b, CR1, CFB, and C5b-9 in kidney tissues and liver was decreased, and the expression of C5 was increased. Network pharmacology screening indicated that AKT, TNF-α, MDM2, UBC, STST3, ESR1, and TP53 were core targets of LNP023 in the treatment of LN. Compared with that in the Normal group, the mRNA expression of the core target in the SLE and LN groups was different; compared with the MRL/lpr group, the LNP023 treatment group showed different mRNA and protein expression levels of AKT, TNF-α, and STST3. ConclusionLNP023 improves LN in MRL/lpr mice. The mechanism is as follows: LNP023 binds to CFB to inhibit the activation of the alternative complement pathway. LNP023 treatment for LN may also play a role in regulating the protein expression of AKT, TNF-α, and STST3.

Complement-Opsonized NIR-IIb Emissive Immunotracers for Dynamically Monitoring Neutrophils in Inflammation-Related Diseases.

Real-time monitoring of neutrophil dynamics is crucial for timely diagnosis and effective treatment of inflammation-related diseases, which requires a reliable tracer for in vivo tracking of neutrophils. However, immunotracers for neutrophils are extremely limited because of the difficulty in labeling the cells. Inspired by the natural biological function of the complement system, a strategy of enhancing the complement C3 opsonization of lanthanide-doped nanoparticles (LnNPs) by modulating their surface chemistry, thus developing a near infrared-IIb emissive nanotracer for neutrophils, is reported herein. Four kinds of surface-modified LnNPs are fabricated, among which phospholipids DOPG-modified LnNPs (LnNPs@PG) with weak antifouling ability and hydroxyl groups adsorb more complement C3 proteins and form covalent linkages with C3b active fragments under inflammation conditions, inducing enhanced complement C3 opsonization. Therefore, LnNPs@PG with enhanced complement C3 opsonization are capable of efficiently labeling inflammation-stimulated neutrophils in vivo through complement-receptors-mediated phagocytosis and achieve dynamic monitoring neutrophils during cutaneous wound healing and cerebral ischemia/reperfusion.

Abnormal lower expression of GPR183 in peripheral blood T and B cell subsets of systemic lupus erythematosus patients

G protein-coupled receptor 183 (GPR183) has been indicated to mediate the migration and localisation of immune cells in T cell-dependent antibody responses. Systemic lupus erythematosus (SLE) is a canonical autoimmune disease involving B cell-mediated tolerance destruction and excessive pathogenic autoantibody production, in which multiple GPCRs play a role. To date, there has been no systematic study regarding the expression of GPR183 in lymphocyte subsets of SLE patients. In this research, firstly, we observed the expression trends of GRP183 in various T and B cell subsets in human tonsil tissues. These lymphocyte subsets include CD4+, CD8+, naïve T, effector T, Tfh, activated Tfh, Th1, Th2, Th17, Treg, CD19+CD27-, CD19+CD27+, naïve B, germinal centre B, memory B, and plasma cells. Further, compared with healthy controls (HCs), GPR183 expression levels in above peripheral blood lymphocyte subsets of patients with SLE were reduced overall. The differential expression of GPR183 expression between inactive and active SLE patients indicates that GPR183 expression may be concerned with the disease activity of SLE. This was further confirmed through the strong negative correlation with SLEDAI score and positive correlation with serum complement protein C3, C4 and C1q levels. Further receiver operating characteristic (ROC) curve analysis revealed that GPR183 expression in circulating CD27-IgD+ B cells may be beneficial in distinguishing between inactive and active SLE patients. In addition, type I interferon stimulation could down-regulate the expression of GPR183 in peripheral blood T and B cell subsets. Aberrant expression of GPR183 may provide some novel insights into disease activity prediction and underlying pathogenesis of SLE.

Practical Management for Use of Eculizumab in the Treatment of Severe, Refractory, Non-Thymomatous, AChR + Generalized Myasthenia Gravis: A Systematic Review.

Myasthenia gravis (MG) is a rare autoimmune disorder caused by specific autoantibodies at the neuromuscular junction. MG is classified by the antigen specificity of these antibodies. Acetylcholine receptor (AChR) antibodies are the most common type (74–88%), followed by anti-muscle specific kinase (MuSK) and other antibodies. While all these antibodies lead to neuromuscular transmission failure, the immuno-pathogenic mechanisms are distinct. Complement activation is a primary driver of AChR antibody-positive MG (AChR+ MG) pathogenesis. This leads to the formation of the membrane attack complex and destruction of AChR receptors and the postsynaptic membrane resulting in impaired neurotransmission and muscle weakness characteristic of MG. Broad-based immune-suppressants like corticosteroids are effective in controlling MG; however, their long-term use can be associated with significant adverse effects. Advances in translational research have led to the development of more directed therapeutic agents that are likely to alter the future of MG treatment. Eculizumab is a humanized monoclonal antibody that inhibits the cleavage of complement protein C5 and is approved for use in generalized MG. In this review, we discuss the pathophysiology of MG; the therapeutic efficacy and tolerability of eculizumab, as well as the practical guidelines for its use in MG; future studies exploring the role of eculizumab in different stages and subtypes of MG subtypes; the optimal duration of therapy and its discontinuation; the characterization of non-responder patients; and the use of biomarkers for monitoring therapy are highlighted. Based on the pathophysiologic mechanisms, emerging therapies and new therapeutic targets are also reviewed.

P840: A MATCHING-ADJUSTED INDIRECT COMPARISON OF THE EFFICACY OF PEGCETACOPLAN USING PRINCE TRIAL DATA VERSUS RAVULIZUMAB AND ECULIZUMAB IN COMPLEMENT-NAÏVE PATIENTS WITH PAROXYSMAL NOCTURNAL HEMOGLOBINURIA

Background: Pegcetacoplan (PEG) is a PEGylated peptide targeting proximal complement protein C3 that has been shown to control both intravascular and extravascular hemolysis and recently approved by the FDA and EMA for the treatment of paroxysmal nocturnal hemoglobinuria (PNH). PRINCE (NCT04085601), was a phase-3, randomized, open-label trial assessing the efficacy and safety of PEG compared to control (CTL) treatment, excluding complement inhibitors among treatment naïve patients with PNH. PEG demonstrated superior efficacy to CTL by improving hemoglobin (Hb) levels and reductions in lactate dehydrogenase (LDH) levels in patients with PNH. To date, no head-to-head clinical trials exist comparing PEG to complement inhibitors in treatment naïve patients. Aims: To assess the comparative effectiveness of PEG for complement-naïve patients with PNH against ravulizumab (RAV) and eculizumab (ECU) using matching-adjusted indirect comparison (MAIC) methodology to conduct cross-trial comparisons. Methods: Individual patient data from PRINCE as well as aggregated published results of RAV and ECU in the ALXN1210-PNH-301 (“301”) study (Lee et al. 2019) were used. To adjust for cross-study differences, propensity score weighting was developed using logistic regression analyses and used to balance demographic and clinical characteristics between the PEG arm and RAV and ECU arms from 301. Outcomes were compared at 26 weeks using unanchored MAIC between PEG versus RAV and PEG versus ECU and included mean and percent change in LDH levels from baseline, LDH normalization (defined as LDH levels dropping <1xULN [246 U/L] in the absence of transfusions during the randomized controlled period), Hb stabilization (defined as the avoidance of a ≥2 g/dL decrease in hemoglobin level in the absence of transfusions during the randomized controlled period), and transfusion avoidance. Weighted Wald tests and 95% confidence intervals were computed for comparisons of categorical and continuous outcomes. Results: A total of 34 patients were included in the PEG arm, 125 patients in the RAV arm, and 121 patients in the ECU arm. Prior to matching, significant differences existed between arms in the proportion of patients with White and American Indian or Alaska Native race, and mean LDH (all p<0.001). After matching the PEG to the RAV and ECU arms, separately, most patient characteristics were well balanced with a mean age of 43.8 years, 44.8 years, 46.2 years, respectively, 43.0-50.0% females, and a mean Hb of 9.4-9.6g/dL across arms; however, there were significant differences in Asian race and baseline LDH (all p<0.01). At week 26, patients in the PEG arm showed a significantly greater improvement in their LDH levels from baseline, with a mean of -523 U/L (-11.2%) compared to RAV and -578 U/L (-12.0%) compared to ECU (all p<0.001; Figure 1). Patients receiving PEG also showed significant improvements in LDH normalization (35.6% compared to RAV, p<0.001; 39.6% compared to ECU, p<0.001), Hb stabilization (28.2% compared to RAV, p<0.001; 31.7% compared to ECU, p<0.001), and transfusion avoidance (22.6% compared to RAV, p<0.001; 30.1% compared to ECU, p<0.001; Figure 1). Image:Summary/Conclusion: In the absence of head-to-head comparisons, this unanchored MAIC study shows that PEG is more efficacious versus RAV and ECU in complement inhibitors treatment naïve PNH patients in terms of significant improvements across all evaluated endpoints, Hb stabilization, LDH improvement, LDH normalization and transfusion avoidance.