Molecular mechanism of complement inhibition by the trypanosome receptor ISG65.

African trypanosomes replicate within infected mammals where they are exposed to the complement system. This system centres around complement C3, which is present in a soluble form in serum but becomes covalently deposited onto the surfaces of pathogens after proteolytic cleavage to C3b. Membrane-associated C3b triggers different complement-mediated effectors which promote pathogen clearance. To counter complement-mediated clearance, African trypanosomes have a cell surface receptor, ISG65, which binds to C3b and which decreases the rate of trypanosome clearance in an infection model. However, the mechanism by which ISG65 reduces C3b function has not been determined. We reveal through cryogenic electron microscopy that ISG65 has two distinct binding sites for C3b, only one of which is available in C3 and C3d. We show that ISG65 does not block the formation of C3b or the function of the C3 convertase which catalyses the surface deposition of C3b. However, we show that ISG65 forms a specific conjugate with C3b, perhaps acting as a decoy. ISG65 also occludes the binding sites for complement receptors 2 and 3, which may disrupt recruitment of immune cells, including B cells, phagocytes, and granulocytes. This suggests that ISG65 protects trypanosomes by combining multiple approaches to dampen the complement cascade.

Nonstructural Protein 1 of Variant PEDV Plays a Key Role in Escaping Replication Restriction by Complement C3.

Zoonotic coronaviruses represent an ongoing threat to public health. The classical porcine epidemic diarrhea virus (PEDV) first appeared in the early 1970s. Since 2010, outbreaks of highly virulent PEDV variants have caused great economic losses to the swine industry worldwide. However, the strategies by which PEDV variants escape host immune responses are not fully understood. Complement component 3 (C3) is considered a central component of the three complement activation pathways and plays a crucial role in preventing viral infection. In this study, we found that C3 significantly inhibited PEDV replication in vitro, and both variant and classical PEDV strains induced high levels of interleukin-1ß (IL-1ß) in Huh7 cells. However, the PEDV variant strain reduces C3 transcript and protein levels induced by IL-1ß compared with the PEDV classical strain. Examination of key molecules of the C3 transcriptional signaling pathway revealed that variant PEDV reduced C3 by inhibiting CCAAT/enhancer-binding protein ß (C/EBP-ß) phosphorylation. Mechanistically, PEDV nonstructural protein 1 (NSP1) inhibited C/EBP-ß phosphorylation via amino acid residue 50. Finally, we constructed recombinant PEDVs to verify the critical role of amino acid 50 of NSP1 in the regulation of C3 expression. In summary, we identified a novel antiviral role of C3 in inhibiting PEDV replication and the viral immune evasion strategies of PEDV variants. Our study reveals new information on PEDV-host interactions and furthers our understanding of the pathogenic mechanism of this virus. IMPORTANCE The complement system acts as a vital link between the innate and the adaptive immunity and has the ability to recognize and neutralize various pathogens. Activation of the complement system acts as a double-edged sword, as appropriate levels of activation protect against pathogenic infections, but excessive responses can provoke a dramatic inflammatory response and cause tissue damage, leading to pathological processes, which often appear in COVID-19 patients. However, how PEDV, as the most severe coronavirus causing diarrhea in piglets, regulates the complement system has not been previously reported. In this study, for the first time, we identified a novel mechanism of a PEDV variant in the suppression of C3 expression, showing that different coronaviruses and even different subtype strains differ in regulation of C3 expression. In addition, this study provides a deeper understanding of the mechanism of the PEDV variant in immune escape and enhanced virulence.

Anti-RNP antibodies are associated with the interferon gene signature but not decreased complement levels in SLE.

OBJECTIVES: The goals of these studies were to elucidate the inter-relationships of specific anti-nuclear antibody (ANA), complement, and the interferon gene signature (IGS) in the pathogenesis of systemic lupus erythematosus (SLE). METHODS: Data from the Illuminate trials were analysed for antibodies to dsDNA as well as RNA-binding proteins (RBP), levels of C3, C4 and various IGS. Statistical hypothesis testing, linear regression analyses and classification and regression trees analysis were employed to assess relationships between the laboratory features of SLE. RESULTS: Inter-relationships of ANAs, complement and the IGS differed between patients of African Ancestry (AA) and European Ancestry (EA); anti-RNP and multiple autoantibodies were more common in AA patients and, although both related to the presence of the IGS, relationships between autoantibodies and complement differed. Whereas, anti-dsDNA had an inverse relationship to C3 and C4, levels of anti-RNP were not related to these markers. The IGS was only correlated with anti-dsDNA in EA SLE and complement was more correlated to the IGS in AA SLE. Finally, autoantibodies occurred in the presence and absence of the IGS, whereas the IGS was infrequent in anti-dsDNA/anti-RBP-negative SLE patients. CONCLUSION: There is a complex relationship between autoantibodies and the IGS, with anti-RNP associated in AA and both anti-dsDNA and RNP associated in EA. Moreover, there was a difference in the relationship between anti-dsDNA, but not anti-RBP, with complement levels. The lack of a relationship of anti-RNP with C3 and C4 suggests that anti-RNP immune complexes (ICs) may drive the IGS without complement fixation, whereas anti-dsDNA ICs involve complement consumption.

Upregulation of Checkpoint Ligand Programmed Death-Ligand 1 in Patients with Paroxysmal Nocturnal Hemoglobinuria Explained by Proximal Complement Activation.

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare hemolytic disease driven by impaired complement regulation. Mutations in genes encoding the enzymes that build the GPI anchors are causative, with somatic mutations in the PIG-A gene occurring most frequently. As a result, the important membrane-bound complement regulators CD55 and CD59 are missing on the affected hematopoietic stem cells and their progeny, rendering those cells vulnerable to complement attack. Immune escape mechanisms sparing affected PNH stem cells from removal are suspected in the PNH pathogenesis, but molecular mechanisms have not been elucidated. We hypothesized that exuberant complement activity in PNH results in enhanced immune checkpoint interactions, providing a molecular basis for the potential immune escape in PNH. In a series of PNH patients, we found increased expression levels of the checkpoint ligand programmed death-ligand 1 (PD-L1) on granulocytes and monocytes, as well as in the plasma of PNH patients. Mechanistically, we demonstrate that complement activation leading to the decoration of particles/cells with C3- and/or C4-opsonins increased PD-L1 expression on neutrophils and monocytes as shown for different in vitro models of classical or alternative pathway activation. We further establish in vitro that complement inhibition at the level of C3, but not C5, inhibits the alternative pathway-mediated upregulation of PD-L1 and show by means of soluble PD-L1 that this observation translates into the clinical situation when PNH patients are treated with either C3 or C5 inhibitors. Together, the presented data show that the checkpoint ligand PD-L1 is increased in PNH patients, which correlates with proximal complement activation.

Marginal zone B cells acquire dendritic cell functions by trogocytosis.

Marginal zone (MZ) B cells produce broad-spectrum antibodies that protect against infection early in life. In some instances, antibody production requires MZ B cells to display pathogen antigens bound to major histocompatibility complex class II (MHC II) molecules to T cells. We describe the trogocytic acquisition of these molecules from conventional dendritic cells (cDCs). Complement component 3 (C3) binds to murine and human MHC II on cDCs. MZ B cells recognize C3 with complement receptor 2 (CR2) and trogocytose the MHC II-C3 complexes, which become exposed on their cell surface. The ubiquitin ligase MARCH1 limits the number of MHC II-C3 complexes displayed on cDCs to prevent their elimination through excessive trogocytosis. Capture of C3 by MHC II thus enables the transfer of cDC-like properties to MZ B cells.

Structure and function of a family of tick-derived complement inhibitors targeting properdin.

Activation of the serum-resident complement system begins a cascade that leads to activation of membrane-resident complement receptors on immune cells, thus coordinating serum and cellular immune responses. Whilst many molecules act to control inappropriate activation, Properdin is the only known positive regulator of the human complement system. By stabilising the alternative pathway C3 convertase it promotes complement self-amplification and persistent activation boosting the magnitude of the serum complement response by all triggers. In this work, we identify a family of tick-derived alternative pathway complement inhibitors, hereafter termed CirpA. Functional and structural characterisation reveals that members of the CirpA family directly bind to properdin, inhibiting its ability to promote complement activation, and leading to potent inhibition of the complement response in a species specific manner. We provide a full functional and structural characterisation of a properdin inhibitor, opening avenues for future therapeutic approaches.

Complement ratios C3bc/C3 and sC5b-9/C5 do not increase the sensitivity of detecting acute complement activation systemically.

BACKGROUND: Complement activation plays an important pathogenic role in numerous diseases. The ratio between an activation product and its parent protein is suggested to be more sensitive to detect complement activation than the activation product itself. In the present study we explored whether the ratio between the activation product and the parent protein for C3 (C3bc/C3) and for C5 (sC5b-9/C5) increased the sensitivity to detect complement activation in acute clinical settings compared to the activation product alone. MATERIALS AND METHODS: Samples from patients with acute heart failure following ST-elevated myocardial infarction (STEMI) and from patients with out-of-hospital cardiac arrest (OHCA) were used. C3, C3bc and C5, sC5b-9 were analysed in 629 and 672 patient samples, respectively. Healthy controls (n = 20) served to determine reference cut-off values for activation products and ratios, defined as two SD above the mean. RESULTS: Increased C3bc/C3- and sC5b-9/C5 ratios were vastly dependent on C3bc and sC5b-9. Thus, 99.5 % and 98.1 % of the increased C3bc/C3- and sC5b-9/C5 ratios were solely dependent on increased C3bc and sC5b-9, respectively. Significantly decreased C3 and C5 caused increased ratios in only 3/600 (0.5 %) and 4/319 (1.3 %) samples, respectively. Strong correlations between C3bc and C3bc/C3-ratio and between sC5b-9 and sC5b-9/C5-ratio were found in the STEMI- (r = 0.926 and r = 0.786, respectively) and the OHCA-population (r = 0.908 and r = 0.843, respectively; p < 0.0001 for all). Importantly, sC5b-9 identified worse outcome groups better than sC5b-9/C5-ratio. CONCLUSION: C3bc and sC5b-9 were sensitive markers of complement activation. The ratios of C3bc/C3 and sC5b-9/C5 did not improve detection of complement activation systemically.

Mesenchymal stem cell therapy attenuates complement C3 deposition and improves the delicate equilibrium between angiogenic and anti-angiogenic factors in abortion-prone mice.

Immunological disorders are one of the main causes of recurrent spontaneous abortions (RSA). A rapidly expanding body of evidence indicates that excessive activation of the complement system is critically involved in the development of miscarriages. In the CBA/J × DBA/2 murine model of recurrent miscarriage, exaggerated and unrestrained complement activation is reported to be the underlying cause of angiogenic factor imbalance and persistent inflammation. We have previously shown that mesenchymal stem cell (MSC) therapy can significantly reduce the abortion rate in abortion-prone mice through regulating the feto-maternal immune response. In the present study, we hypothesized that MSCs might improve the balance of angiogenic factors at the feto-maternal unit of CBA/J × DBA/2 mice by restraining complement activation and deposition. To explore this hypothesis, autologous adipose tissue-derived mesenchymal stem cells (AD-MSCs) were administered intra-peritoneally to abortion-prone mice on the 4.5th day of gestation. Control mice received PBS as vehicle. On day 13.5 of pregnancy, deposition of the complement component C3 and expression levels of Crry, CFD (adipsin), VEGF, PlGF and FLT-1 were measured at the feto-maternal interface by immunohistochemistry and real-time PCR, respectively. Decidual cells were also cultured in RPMI 1640 medium for 48 h and VEGF and sFLT-1 protein levels were quantified in supernatants using enzyme-linked immunosorbent assay (ELISA). Our results indicated that MSC therapy significantly reduced C3 deposition and adipsin transcription in the fetal-maternal interface of abortion-prone mice. Furthermore, administration of MSCs robustly upregulated the mRNA expression levels of Crry, VEGF, PlGF and FLT-1 in the placenta and decidua of CBA/J × DBA/2 mice. Consistently, the in vitro results demonstrated that decidual cells obtained from MSC-treated dams produced increased concentrations of VEGF in culture supernatants, with concomitant decreased levels of sFLT-1 protein. Here, we show for the first time that adoptive transfer of MSCs rectifies the disturbed balance of angiogenic factors observed at the feto-maternal unit of CBA/J × DBA/2 mice, in part at least, through inhibiting excessive complement activation and promoting the production of angiogenic factors. Collectively, these alterations seem to play a pivotal role in reducing the abortion rate and improving the intrauterine condition for the benefit of the fetus.

Homodimeric Minimal Factor H: In Vivo Tracking and Extended Dosing Studies in Factor H Deficient Mice.

C3 glomerulopathy (C3G) is associated with dysregulation of the alternative pathway (AP) of complement and treatment options remain inadequate. Factor H (FH) is a potent regulator of the AP. An in-depth analysis of FH-related protein dimerised minimal (mini)-FH constructs has recently been published. This analysis showed that addition of a dimerisation module to mini-FH not only increased serum half-life but also improved complement regulatory function, thus providing a potential treatment option for C3G. Herein, we describe the production of a murine version of homodimeric mini-FH [mHDM-FH (mFH1-5^18-20^R1-2)], developed to reduce the risk of anti-drug antibody formation during long-term experiments in murine models of C3G and other complement-driven pathologies. Our analysis of mHDM-FH indicates that it binds with higher affinity and avidity to WT mC3b when compared to mouse (m)FH (mHDM-FH KD=505 nM; mFH KD=1370 nM) analogous to what we observed with the respective human proteins. The improved binding avidity resulted in enhanced complement regulatory function in haemolytic assays. Extended interval dosing studies in CFH-/- mice (5mg/kg every 72hrs) were partially effective and bio-distribution analysis in CFH-/- mice, through in vivo imaging technologies, demonstrates that mHDM-FH is preferentially deposited and remains fixed in the kidneys (and liver) for up to 4 days. Extended dosing using an AAV- human HDM-FH (hHDM-FH) construct achieved complete normalisation of C3 levels in CFH-/- mice for 3 months and was associated with a significant reduction in glomerular C3 staining. Our data demonstrate the ability of gene therapy delivery of mini-FH constructs to enhance complement regulation in vivo and support the application of this approach as a novel treatment strategy in diseases such as C3G.

Complement Factor H-Related 3 Enhanced Inflammation and Complement Activation in Human RPE Cells.

Complement Factor H-Related 3 (FHR-3) is a major regulator of the complement system, which is associated with different diseases, such as age-related macular degeneration (AMD). However, the non-canonical local, cellular functions of FHR-3 remained poorly understood. Here, we report that FHR-3 bound to oxidative stress epitopes and competed with FH for interaction. Furthermore, FHR-3 was internalized by viable RPE cells and modulated time-dependently complement component (C3, FB) and receptor (C3aR, CR3) expression of human RPE cells. Independently of any external blood-derived proteins, complement activation products were detected. Anaphylatoxin C3a was visualized in treated cells and showed a translocation from the cytoplasm to the cell membrane after FHR-3 exposure. Subsequently, FHR-3 induced a RPE cell dependent pro-inflammatory microenvironment. Inflammasome NLRP3 activation and pro-inflammatory cytokine secretion of IL-1ß, IL-18, IL-6 and TNF-α were induced after FHR-3-RPE interaction. Our previously published monoclonal anti-FHR-3 antibody, which was chimerized to reduce immunogenicity, RETC-2-ximab, ameliorated the effect of FHR-3 on ARPE-19 cells. Our studies suggest FHR-3 as an exogenous trigger molecule for the RPE cell "complosome" and as a putative target for a therapeutic approach for associated degenerative diseases.

A novel C-type lectin activates the complement cascade in the primitive oyster Crassostrea gigas.

The ancient origin of the lectin pathway of the complement system can be traced back to protochordates (such as amphioxus and tunicates) by the presence of components such as ficolin, glucose-binding lectin, mannose-binding lectin-associated serine protease (MASP), and C3. Evidence for a more primitive origin is offered in the present study on the Pacific oyster Crassostrea gigas. C3 protein in C. gigas (CgC3) was found to be cleaved after stimulation with the bacteria Vibrio splendidus. In addition, we identified a novel C-type lectin (defined as CgCLec) with a complement control protein (CCP) domain, which recognized various pathogen-associated molecular patterns (PAMPs) and bacteria. This protein was involved in the activation of the complement system by binding CgMASPL-1 to promote cleavage of CgC3. The production of cytokines and antibacterial peptides, as well as the phagocytotic ratio of haemocytes in CgCLec-CCP-, CgMASPL-1-, or CgC3-knockdown oysters, decreased significantly after V. splendidus stimulation. Moreover, this activated CgC3 participated in perforation of bacterial envelopes and inhibiting survival of the infecting bacteria. These results collectively suggest that there existed an ancient lectin pathway in molluscs, which was activated by a complement cascade to regulate the production of immune effectors, phagocytosis, and bacterial lysis.

Lung mesenchymal stromal cells influenced by Th2 cytokines mobilize neutrophils and facilitate metastasis by producing complement C3.

Pre-metastatic niche formation is critical for the colonization of disseminated cancer cells in distant organs. Here we find that lung mesenchymal stromal cells (LMSCs) at pre-metastatic stage possess potent metastasis-promoting activity. RNA-seq reveals an upregulation of complement 3 (C3) in those LMSCs. C3 is found to promote neutrophil recruitment and the formation of neutrophil extracellular traps (NETs), which facilitate cancer cell metastasis to the lungs. C3 expression in LMSCs is induced and sustained by Th2 cytokines in a STAT6-dependent manner. LMSCs-driven lung metastasis is abolished in Th1-skewing Stat6-deficient mice. Blockade of IL-4 by antibody also attenuates LMSCs-driven cancer metastasis to the lungs. Consistently, metastasis is greatly enhanced in Th2-skewing T-bet-deficient mice or in nude mice adoptively transferred with T-bet-deficient T cells. Increased C3 levels are also detected in breast cancer patients. Our results suggest that targeting the Th2-STAT6-C3-NETs cascade may reduce breast cancer metastasis to the lungs.

Recognition of Tumor-Associated Antigens and Immune Subtypes in Glioma for mRNA Vaccine Development.

Background: Although mRNA vaccines have been efficient for combating a variety of tumors, their effectiveness against glioma remains unclear. There is growing evidence that immunophenotyping can reflect the comprehensive immune status and microenvironment of the tumor, which correlates closely with treatment response and vaccination potency. The purpose of this research was to screen for effective antigens in glioma that could be used for developing mRNA vaccines and to further differentiate the immune subtypes of glioma to create an selection criteria for suitable patients for vaccination. Methods: Gene expression profiles and clinical data of 698 glioma samples were extracted from The Cancer Genome Atlas, and RNA_seq data of 1018 glioma samples was gathered from Chinese Glioma Genome Atlas. Gene Expression Profiling Interactive Analysis was used to determine differential expression genes and prognostic markers, cBioPortal software was used to verify gene alterations, and Tumor Immune Estimation Resource was used to investigate the relationships among genes and immune infiltrating cells. Consistency clustering was applied for consistent matrix construction and data aggregation, Gene oncology enrichment was performed for functional annotation, and a graph learning-based dimensionality reduction method was applied to describe the subtypes of immunity. Results: Four overexpressed and mutated tumor antigens associated with poor prognosis and infiltration of antigen presenting cells were identified in glioma, including TP53, IDH1, C3, and TCF12. Besides, four immune subtypes of glioma (IS1-IS4) and 10 immune gene modules were identified consistently in the TCGA data. The immune subtypes had diverse molecular, cellular, and clinical features. IS1 and IS4 displayed an immune-activating phenotype and were associated with worse survival than the other two subtypes, while IS2 and IS3 had lower levels of tumor immune infiltration. Immunogenic cell death regulators and immune checkpoints were also diversely expressed in the four immune subtypes. Conclusion: TP53, IDH1, C3, and TCF12 are effective antigens for the development of anti-glioma mRNA vaccines. We found four stable and repeatable immune subtypes of human glioma, the classification of the immune subtypes of glioma may play a crucial role in the predicting mRNA vaccine outcome.

Phase IIa clinical trial of complement C3 inhibitor AMY-101 in adults with periodontal inflammation.

BackgroundGingivitis and periodontitis are prevalent inflammatory diseases of the periodontal tissues. Current treatments are often ineffective or do not prevent disease recurrence. Uncontrolled complement activation and the resulting chronic gingival inflammation are hallmarks of periodontal diseases. We determined the efficacy and safety of a complement 3-targeted therapeutic, AMY-101, which was locally administered to adult patients with periodontal inflammation.MethodsThirty-two patients with gingival inflammation were enrolled in a randomized, placebo-controlled, double-blind, split-mouth phase IIa trial that followed a dose escalation study to select a safe and effective dose in an additional 8 patients. Half of the patient's mouth was randomly assigned to AMY-101 (0.1 mg/site) or placebo injections at sites of inflammation, administered on days 0, 7, and 14, and then evaluated for safety and efficacy outcomes on days 28, 60, and 90. The primary efficacy outcome was a change in gingival inflammation, measured by a modified gingival index (MGI), and secondary outcomes included changes in bleeding on probing (BOP), the amount of plaque, pocket depth, clinical attachment level, and gingival crevicular fluid levels of matrix metalloproteinases (MMPs) over 90 days.ResultsA once-weekly intragingival injection of AMY-101 for 3 weeks was safe and well tolerated in all participants and resulted in significant (P < 0.001) reductions in clinical indices measuring gingival inflammation (MGI and BOP). AMY-101 significantly (P < 0.05) reduced MMP-8 and MMP-9 levels, indicators of inflammatory tissue destruction. These therapeutic effects persisted for at least 3 months after treatment.ConclusionAMY-101 treatment resulted in a significant and sustainable reduction in gingival inflammation without adverse events and, we believe, merits further investigation for the treatment of periodontitis and other oral or peri-implant inflammatory conditions.Trial registrationClinicalTrials.gov identifier NCT03694444.FundingAmyndas Pharmaceuticals.

FHR-5 Serum Levels and CFHR5 Genetic Variations in Patients With Immune Complex-Mediated Membranoproliferative Glomerulonephritis and C3-Glomerulopathy.

Background: Factor H-related protein 5 (FHR-5) is a member of the complement Factor H protein family. Due to the homology to Factor H, the main complement regulator of the alternative pathway, it may also be implicated in the pathomechanism of kidney diseases where Factor H and alternative pathway dysregulation play a role. Here, we report the first observational study on CFHR5 variations along with serum FHR-5 levels in immune complex-mediated membranoproliferative glomerulonephritis (IC-MPGN) and C3 glomerulopathy (C3G) patients together with the clinical, genetic, complement, and follow-up data. Methods: A total of 120 patients with a histologically proven diagnosis of IC-MPGN/C3G were enrolled in the study. FHR-5 serum levels were measured in ELISA, the CFHR5 gene was analyzed by Sanger sequencing, and selected variants were studied as recombinant proteins in ELISA and surface plasmon resonance (SPR). Results: Eight exonic CFHR5 variations in 14 patients (12.6%) were observed. Serum FHR-5 levels were lower in patients compared to controls. Low serum FHR-5 concentration at presentation associated with better renal survival during the follow-up period; furthermore, it showed clear association with signs of complement overactivation and clinically meaningful clusters. Conclusions: Our observations raise the possibility that the FHR-5 protein plays a fine-tuning role in the pathogenesis of IC-MPGN/C3G.

C3 complement inhibition prevents antibody-mediated rejection and prolongs renal allograft survival in sensitized non-human primates.

Sensitized kidney transplant recipients experience high rates of antibody-mediated rejection due to the presence of donor-specific antibodies and immunologic memory. Here we show that transient peri-transplant treatment with the central complement component C3 inhibitor Cp40 significantly prolongs median allograft survival in a sensitized nonhuman primate model. Despite donor-specific antibody levels remaining high, fifty percent of Cp40-treated primates maintain normal kidney function beyond the last day of treatment. Interestingly, presence of antibodies of the IgM class associates with reduced median graft survival (8 vs. 40 days; p = 0.02). Cp40 does not alter lymphocyte depletion by rhesus-specific anti-thymocyte globulin, but inhibits lymphocyte activation and proliferation, resulting in reduced antibody-mediated injury and complement deposition. In summary, Cp40 prevents acute antibody-mediated rejection and prolongs graft survival in primates, and inhibits T and B cell activation and proliferation, suggesting an immunomodulatory effect beyond its direct impact on antibody-mediated injury.

Scientific memory from the early nineties; a common project with professors late János Gergely and Anna Erdei.

Based on the findings of common project 29 years ago, the Scandinavian J. of Immunology accepted and published our paper entitled by "FcγR-Dependent Regulation of the Biosynthesis of Complement C3 by Murine Macrophages: the Modulatory Effect of IL-6" (Bajtay et al. in SJI 35:195-201, 1992). In this report we attempt to review the previous results and evaluate them with our current concepts on the interaction between the actors of adaptive and innate immunity. Let us first to summarize the basic results and consequences from the paper from 1992. Abstract from 1991-1992: The effect of murine IgG isotypes (myeloma proteins) on the gene expression and secretion of the third component of complement (C3) has been studied using the in monocytoid cell line P388D1 and oil-elicited mouse peritoneal macrophages. It is demonstrated that the binding of lgG2a and lgG2b but not IgGl and IgG3 isotypes augments the biosynthesis of C3 both in the presence and in the absence of the phorbol myristate acetate in the case of both cell types. The multifunctional cytokine inlerleukin-6 (IL-6) alone reveals no effect on the gene expression of C3, but facilitates the effectiveness of mouse IgG2a and IgG2b. Confirming the role of FcgRll, a strong up-regulation of gene expression and secretion of C3 was found when macrophages were co-cultured with the F(ab')2 fragment of the FcγRII-specific monoclonal antibody 2.4 G2.

Different Aspects of Classical Pathway Overactivation in Patients With C3 Glomerulopathy and Immune Complex-Mediated Membranoproliferative Glomerulonephritis.

The rare and heterogeneous kidney disorder C3 glomerulopathy (C3G) is characterized by dysregulation of the alternative pathway (AP) of the complement system. C3G is often associated with autoantibodies stabilizing the AP C3 convertase named C3 nephritic factors (C3NeF). The role of classical pathway (CP) convertase stabilization in C3G and related diseases such as immune complex-mediated membranoproliferative glomerulonephritis (IC-MPGN) remains largely unknown. Here, we investigated the CP convertase activity in patients with C3G and IC-MPGN. Using a refined two-step hemolytic assay, we measured the stability of CP convertases directly in the serum of 52 patients and 17 healthy controls. In four patients, CP convertase activity was prolonged compared to healthy controls, i.e. the enzymatic complex was stabilized. In three patients (2 C3G, 1 IC-MPGN) the convertase stabilization was caused by immunoglobulins, indicating the presence of autoantibodies named C4 nephritic factors (C4NeFs). Importantly, the assay also enabled detection of non-immunoglobulin-mediated stabilization of the CP convertase in one patient with C3G. Prolonged CP convertase activity coincided with C3NeF activity in all patients and for up to 70 months of observation. Crucially, experiments with C3-depleted serum showed that C4NeFs stabilized the CP C3 convertase (C4bC2a), that does not contain C3NeF epitopes. All patients with prolonged CP convertase activity showed clear signs of complement activation, i.e. lowered C3 and C5 levels and elevated levels of C3d, C3bc, C3bBbP, and C5b-9. In conclusion, this work provides new insights into the diverse aspects and (non-)immunoglobulin nature of factors causing CP convertase overactivity in C3G/IC-MPGN.

Insights Into the Structure-Function Relationships of Dimeric C3d Fragments.

Cleavage of C3 to C3a and C3b plays a central role in the generation of complement-mediated defences. Although the thioester-mediated surface deposition of C3b has been well-studied, fluid phase dimers of C3 fragments remain largely unexplored. Here we show C3 cleavage results in the spontaneous formation of C3b dimers and present the first X-ray crystal structure of a disulphide-linked human C3d dimer. Binding studies reveal these dimers are capable of crosslinking complement receptor 2 and preliminary cell-based analyses suggest they could modulate B cell activation to influence tolerogenic pathways. Altogether, insights into the physiologically-relevant functions of C3d(g) dimers gained from our findings will pave the way to enhancing our understanding surrounding the importance of complement in the fluid phase and could inform the design of novel therapies for immune system disorders in the future.

The Contribution of Serum Complement Component 3 Levels to 90-Day Mortality in Living Donor Liver Transplantation.

The contributions of the complement system have been elucidated in the process of solid organ transplantation, including kidney transplantation. However, the role of complement in liver transplantation is unknown. We sought to elucidate the time-dependent changes of peritransplantational serum complement levels and the relationships with posttransplant outcomes and other immunological biomarkers. We enrolled 82 patients who underwent living-related donor liver transplantation (LDLT). Nine patients (11%) died within 90 days after LDLT (non-survivors). The following immunomarkers were collected preoperatively and at 1, 2, and 4 week(s) after LDLT: serum C3, C4, immunoglobulin G (IgG), and peripheral blood leukocyte populations characterized by CD3, CD4, CD8, CD16, CD19, CD20, CD22, and CD56. Consequently, C3 and C4 increased time-dependently after LDLT. Preoperatively, C3 was negatively correlated with the MELD score, Child-Pugh score, CD16-positive leukocyte percentage, and the CD56-positive leukocyte percentage. Non-survivors had lower levels of C3 at 2 weeks in comparison to survivors (median [interquartile range]: 56 [49-70] mg/dL vs. 88 [71-116] md/dL, p=0.0059). When the cutoff value of C3 at 2 weeks to distinguish non-survivors was set to 71 mg/dL, the sensitivity, specificity, and area under the ROC curve were 87.5%, 75.0%, and 0.80, respectively. A principal component analysis showed an inverse relationship between the C3 and C4 levels and the percentage of CD8-, CD16-, and CD56-positive leukocytes at 1 and 2 week(s). All non-survivors were included in the cluster that showed higher percentages of CD8-, CD16-, and CD56-positive leukocytes at 2 weeks. In conclusion, we demonstrated the relationship between complement, outcomes, and other immunomarkers in LDLT and suggested the usefulness of C3 at 2 weeks after LDLT in distinguishing the mortality.