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.

Modular complement assemblies for mitigating inflammatory conditions.

Complement protein C3dg, a key linkage between innate and adaptive immunity, is capable of stimulating both humoral and cell-mediated immune responses, leading to considerable interest in its use as a molecular adjuvant. However, the potential of C3dg as an adjuvant is limited without ways of controllably assembling multiple copies of it into vaccine platforms. Here, we report a strategy to assemble C3dg into supramolecular nanofibers with excellent compositional control, using ß-tail fusion tags. These assemblies were investigated as therapeutic active immunotherapies, which may offer advantages over existing biologics, particularly toward chronic inflammatory diseases. Supramolecular assemblies based on the Q11 peptide system containing ß-tail-tagged C3dg, B cell epitopes from TNF, and the universal T cell epitope PADRE raised strong antibody responses against both TNF and C3dg, and prophylactic immunization with these materials significantly improved protection in a lethal TNF-mediated inflammation model. Additionally, in a murine model of psoriasis induced by imiquimod, the C3dg-adjuvanted nanofiber vaccine performed as well as anti-TNF monoclonal antibodies. Nanofibers containing only ß-tail-C3dg and lacking the TNF B cell epitope also showed improvements in both models, suggesting that supramolecular C3dg, by itself, played an important therapeutic role. We observed that immunization with ß-tail-C3dg caused the expansion of an autoreactive C3dg-specific T cell population, which may act to dampen the immune response, preventing excessive inflammation. These findings indicate that molecular assemblies displaying C3dg warrant further development as active immunotherapies.

Intensity of de novo DSA detected by Immucor Lifecodes assay and C3d fixing antibodies are not predictive of subclinical ABMR after Kidney Transplantation.

De novo donor-specific antibodies (dnDSA) are associated with antibody-mediated rejection (ABMR) and allograft loss. We tested Immucor* (IM) Luminex Single-antigen beads (LSAB) assay and C3d-fixing antibodies in the setting of dnDSA and subclinical (s) ABMR. This retrospective multicentric study included 123 patients biopsied because of the presence of subclinical de novo DSA detected by One Lamda* Labscreen (MFI > 1000). In 112 patients, sera of the day of the biopsy were available and tested in a central lab with IM Lifecodes LSAB and C3d fixing antibodies assays. In 16 patients (14.3%), no DSA was detected using Immucor test. In 96 patients, at least one DSA was determined with IM. Systematic biopsies showed active sABMR in 30 patients (31.2%), chronic active sABMR in 17 patients (17.7%) and no lesions of sABMR in 49 KT recipients (51%). Intensitity criteria (BCM, BCR and AD-BCR) of DSA were not statistically different between these 3 histological groups. The proportion of patients with C3d-fixing DSA was not statistically different between the 3 groups and did not offer any prognostic value regarding graft survival. Performing biopsy for dnDSA could not be guided by the intensity criteria of IM LSAB assay. C3d-fixing DSA do not offer added value.

Cloning and structural analysis of complement component 3d in wild birds provides insight into its functional evolution.

Complement component 3 d (C3d) is the final cleavage product of the complement component C3 and serves as a crucial role in link innate and adaptive immunity, and increase B-cell sensitivity to an antigen by 1000-10000 fold. The crystal structure of human C3d revealed there are two distinct surfaces, a convex surface containing the thioester-constituting residues that mediate covalent binding to the target antigen, and a concave surface with an acidic pocket responsible for interaction with CR2. In this study, we cloned and sequenced cDNA fragment encoding C3d region from 15 wild bird species. Then, the C3d sequences from wild birds, chicken and mammals were aligned to construct phylogenetic trees. Phylogenetic tree displayed two main branches, indicating mammals and birds, but the bird C3d branch was divided into two main parts, with five wild birds (Ardeola bacchus, Zoothera, Bubo, Crossoptilon mantchuricum and Caprimulgus europaeus) clustering much closer to mammals. In addition, the C3d proteins of Ardeola bacchus, Bubo, Crossoptilon mantchuricum and Caprimulgus europaeus contained a Glu163 residue at the position at which Lys163 was found in other birds. However, Glu163 have the same charge polarity as Asp163, which is the key amino acid residue comprising the acidic pocket combined with CR2 found at this position in mammals, and Zoothera also possessed Asp163 at this position. Structure modeling analyses also verified that the C3ds of these five wild bird species exhibited the amino acid sequence and structure comprising the typical acidic pocket found in mammals that is required for combination with B cell surface receptors, which contribute electrostatic forces to interact with CR2. Our investigations indicate that some bird C3ds may already have the ability to bind with CR2 by electrostatic force, like mammals. As Ardeola bacchus, Zoothera, Bubo, Crossoptilon mantchuricum and Caprimulgus europaeus have more typical C3d concave acid pockets and thus a stronger ability to bind CR2, we speculate that these five wild birds may have a solider immunity against pathogens. Our phylogenetic and structural analyses of bird C3ds provide insights on the evolutionary divergence in the function of immune factors of avian and mammalian.

Does a Useful Test Exist to Properly Evaluate the Pathogenicity of Donor-specific Antibodies? Lessons From a Comprehensive Analysis in a Well-studied Single-center Kidney Transplant Cohort.

BACKGROUND: Donor-specific antibodies (DSA) play a major role in antibody-mediated rejection (AMR) and graft dysfunction. However, the clinical relevance of complement-binding anti-HLA antibodies remains unclear. METHODS: Here, we analyzed DSA detected in the serum (sDSA) using single antigen bead, C1q, and C3d assays combined with the study of intragraft DSA (gDSA) in 86 patients who had DSA and underwent a kidney biopsy for cause (n = 58) or without evidence of kidney dysfunction (n = 28). DSA characteristics were collected and related to the presence of AMR, graft histological features, and allograft survival. RESULTS: Forty-five patients (52%) had C1q DSA, and 42 (51%) had C3d DSA. Allograft biopsies revealed AMR in 63 cases (73%), regardless of kidney function. gDSA were identified in 74% of biopsies. We observed a strong correlation among single antigen bead mean fluorescence intensity and complement assays positivity, presence of gDSA, and AMR occurrence. CONCLUSIONS: Complement-binding DSA per se were not significantly associated with allograft survival in the entire study sample. Finally, gDSA predicted subsequent graft loss in patients who showed a stable renal function at the day of biopsy. Our data suggest that DSA mean fluorescence intensity and presence of gDSA might provide prognostic information during posttransplant monitoring.

Targeting the Immune Complex-Bound Complement C3d Ligand as a Novel Therapy for Lupus.

Humoral autoimmunity is central to the development of systemic lupus erythematosus (SLE). Complement receptor type 2 (CR2)/CD21 plays a key role in the development of high-affinity Abs and long-lasting memory to foreign Ags. When CR2 is bound by its primary C3 activation fragment-derived ligand, designated C3d, it coassociates with CD19 on B cells to amplify BCR signaling. C3d and CR2 also mediate immune complex binding to follicular dendritic cells. As the development of SLE involves subversion of normal B cell tolerance checkpoints, one might expect that CR2 ligation by C3d-bound immune complexes would promote development of SLE. However, prior studies in murine models of SLE using gene-targeted Cr2-/- mice, which lack both CR2 and complement receptor 1 (CR1), have demonstrated contradictory results. As a new approach, we developed a highly specific mouse anti-mouse C3d mAb that blocks its interaction with CR2. With this novel tool, we show that disruption of the critical C3d-CR2 ligand-receptor binding step alone substantially ameliorates autoimmunity and renal disease in the MRL/lpr model of SLE.

A candidate DNA vaccine encoding a fusion protein of porcine complement C3d-P28 and ORF2 of porcine circovirus type 2 induces cross-protective immunity against PCV2b and PCV2d in pigs.

BACKGROUND: Porcine circovirus type 2 (PCV2) is an economically important viral pathogen for swine industry worldwide. However, current PCV2 vaccines provide incomplete protection against the PCV2d, which has recently emerged as the predominant pathogenic form of PCV2. METHODS: To develop a novel DNA vaccine with high efficacy against PCV2d virus, we fused the ORF2 of PCV2d to three copies of the minimum-binding domain of the complement C3 cascade terminal component, C3d-P28. Expression of ORF2 alone (pVO) or fused C3d-P28 (pVOC3) were verified by immunofluorescent assay. Vaccine efficacy was tested by measured the DNA copy and T and B cell immune response. RESULTS: Vaccination with pVOC3 reduced the levels of PCV2 genomic DNA after pigs were infected with either PCV2b or PCV2d genotypes, produced potent antibodies against PCV2, and stimulated PCV2-specific interferon-γ secreting cells. CONCLUSION: Results suggested pVOC3 would be a safe and effective DNA vaccine to confer cross-protection against both PCV2b and PCV2d genotypes in pigs.

Getting on target: Development of the novel, prozone-resistant, dual antibody rapid test (DART) for the LABScreen single antigen bead (SAB) assay.

A major limitation of the single antigen bead (SAB) assay is the so called prozone effect, whereby the detection of high titer complement fixing HLA antibodies is compromised due to complement split product (from C3 and C4 components) deposition and interference with the reporter anti-IgG-PE antibody binding. Strategies to minimize prozone include serum titration or treatment with heat, dithiotreitol (DTT), or ethylenediaminetetraacetic acid (EDTA). While effective, these treatments may compromise HLA antibody binding and detection. Here we describe the Dual Antibody Rapid Test (DART), a modified version of the rapid optimized SAB (ROB) protocol, in which we use an IgG-PE/C3d-PE antibody cocktail to simultaneously detect bead bound IgG and C3d, which allows for detection of HLA antibodies independent of the prozone effect. Twenty prozone positive sera (10 class I and 10 class II), identified by titration, were tested by the ROB protocol, with or without EDTA pre-treatment, using three reporter antibody cocktails: (1) IgG-PE, (2) C3d-PE, or (3) IgG-PE/C3d-PE (DART). Mean fluorescence intensity (MFI) values were then compared. IgG negative (n = 735) vs IgG positive (n = 1185) reactions were identified using a 1000 MFI IgG EDTA cutoff. IgG positive reactions were classified based on ΔMFI (IgG EDTA - IgG) as follows: (1) prozone negative (ΔMFI < 3000; n = 737), (2) slight prozone (ΔMFI 3001-5000; n = 49), (3) moderate prozone (ΔMFI 5001-10,000; n = 93), and (4) marked prozone (ΔMFI > 10,001; n = 306). No C3d deposition was present on IgG negative beads, and the majority of prozone positive specificities (438/448; 98%) fixed complement and were detected with the C3d-PE reporter. Interestingly, C3d-PE MFI was directly proportional to the degree of prozone (mean C3d-PE MFI = 4419.5 ±â€¯1606.3 for slight, 5991.0 ±â€¯2302.7 for moderate, and 12,417.4 ±â€¯2969.9 for marked prozone specificities). Interestingly, EDTA treatment was found to have a negative impact on MFI of up to 15% of prozone negative specificities. Importantly, the DART protocol detected all prozone positive specificities while MFI for prozone negative specificities correlated well with those seen with the IgG-PE reporter alone (R2 = 0.97). In conclusion, the DART protocol accurately detects HLA antibodies independent of the prozone effect. Implementation of DART is an easy way to overcome the prozone effect without compromising HLA antibody detection.

The ABCs (DRDQDPs) of the prozone effect in single antigen bead HLA antibody testing: Lessons from our highly sensitized patients.

Accurate identification of HLA antibodies using the single antigen bead (SAB) assay is critical for assessment of pre/post-transplant immunological risk and successful virtual crossmatching. Unfortunately, high titer HLA antibodies can be missed or underestimated in the SAB assay as a result of interference with the detection of IgG. This so called prozone effect has been attributed to both complement- and IgM-dependent mechanisms and can be minimized with serum dilution or treatment with heat, EDTA, or DTT. In this study we describe the frequency, nature, and degree of prozone in a cohort of highly sensitized patients (cPRA ≥ 95%), in whom accurate detection of HLA antibodies and virtual crossmatching is of paramount importance. Sera were tested by the SAB assay ±â€¯EDTA treatment, ±1:10 dilution to identify the prozone effect. The relative contribution of complement vs IgM to prozone was assessed using anti-C3d and anti-IgM reporter antibodies, respectively. We found that prozone was very frequent in highly sensitized patients (80%), especially those with a history of previous transplantation (87%). Class I HLA specificities were more commonly affected than class II and the susceptibility to prozone was locus dependent with HLA-A(31%), -B(29%) and -DQ(26%) being affected more frequently than HLA-DP(17%), -C(16%) and -DR(5%) antigens. Interestingly, the presence of prozone could be predicted by C3d positivity (MFI ≥ 4000; sensitivity = 95.2%, specificity = 97.2%) and the degree of prozone correlated directly with the extent of C3d deposition. The role of IgM was less clear. However, serum dilution studies suggested that IgM may contribute to interference in a small subset of prozone positive specificities. Our study underscores the importance of serum treatment to inhibit complement activation and minimize prozone in the SAB assay, especially in highly sensitized patients.

Differential immunoglobulin and complement levels in leprosy prior to development of reversal reaction and erythema nodosum leprosum.

BACKGROUND: Leprosy is a treatable infectious disease caused by Mycobacterium leprae. However, there is additional morbidity from leprosy-associated pathologic immune reactions, reversal reaction (RR) and erythema nodosum leprosum (ENL), which occur in 1 in 3 people with leprosy, even with effective treatment of M. leprae. There is currently no predictive marker in use to indicate which people with leprosy will develop these debilitating immune reactions. Our peripheral blood mononuclear cell (PBMC) transcriptome analysis revealed that activation of the classical complement pathway is common to both RR and ENL. Additionally, differential expression of immunoglobulin receptors and B cell receptors during RR and ENL support a role for the antibody-mediated immune response during both RR and ENL. In this study, we investigated B-cell immunophenotypes, total and M. leprae-specific antibodies, and complement levels in leprosy patients with and without RR or ENL. The objective was to determine the role of these immune mediators in pathogenesis and assess their potential as biomarkers of risk for immune reactions in people with leprosy. METHODOLOGY/FINDINGS: We followed newly diagnosed leprosy cases (n = 96) for two years for development of RR or ENL. They were compared with active RR (n = 35), active ENL (n = 29), and healthy household contacts (n = 14). People with leprosy who subsequently developed ENL had increased IgM, IgG1, and C3d-associated immune complexes with decreased complement 4 (C4) at leprosy diagnosis. People who developed RR also had decreased C4 at leprosy diagnosis. Additionally, elevated anti-M. leprae antibody levels were associated with subsequent RR or ENL. CONCLUSIONS: Differential co-receptor expression and immunoglobulin levels before and during immune reactions intimate a central role for humoral immunity in RR and ENL. Decreased C4 and elevated anti-M. leprae antibodies in people with new diagnosis of leprosy may be risk factors for subsequent development of leprosy immune reactions.

Systematic Screening Using Luminex for De Novo C3d Fixing of Class II Donor-Specific Antibodies Is Correlated With Luminex Mean Fluorescence Intensity in Renal Transplant Patients.

OBJECTIVES: Chronic antibody-mediated rejection is the main cause of late kidney graft loss. The presence of donor-specific antibodies in the serum is the main criterion for this diagnosis. Single antigen Luminex assays can identify donor-specific antibodies, and semiquantitative estimates of antibodies can be assessed using mean fluorescence intensity. Recent data have shown that patients whose donor-specific antibodies fix C3d have worse clinical outcomes, implying that C3d-specific Luminex assays may provide useful prognostic data. MATERIALS AND METHODS: We compared C3d donor-specific antibodies with standard immunoglobulin G donor-specific antibody mean fluorescence intensities in a cohort of patients with de novo class II donor-specific antibodies and analyzed subsequent graft survival. The included kidney graft recipients received transplants between 2005 and 2015 and had developed de novo class II donor-specific antibodies. Serum was tested using the standard single antigen Luminex technique and the C3d-fixing antibody-detection system (Immucor, Herentals, Belgium). RESULTS: In our patient cohort, 41/924 patients (4.4%) developed class II donor-specific antibodies, and 65 serum samples were analyzed (at baseline and follow-up). Among these samples, 43 (66%) were negative for C3d donor-specific antibodies. A mean fluorescence intensity threshold of 9000 in the single antigen Luminex assay discerned all negative (from positive) C3d donor-specific antibodies, even when all single-bead results were taken into account. Sixteen patients (39%) had poor outcomes (ie, either creatinine levels had doubled or they had lost their graft) over the median follow-up of 5 years. C3d results were significantly associated with graft survival (P = .04). We found a strong correlation between C3d-fixing antibody positivity and mean fluorescence intensity strength in the setting of de novo class II donor-specific antibodies. CONCLUSIONS: These results further reinforce the paradigm that the higher the mean fluorescence intensity, the more complement activation occurs. Routine C3d testing is thus unnecessary in this setting.

C3d-binding Donor-specific HLA Antibody Is Associated With a High Risk of Antibody-mediated Rejection and Graft Loss in Stable Kidney Transplant Recipients: A Single-center Cohort Study.

BACKGROUND: One risk factor for antibody-mediated rejection (ABMR) and poor outcome after kidney transplantation is donor-specific anti‒human leukocyte antigen (anti-HLA) antibodies (DSAs). In this study we sought to determine whether the presence of DSAs that bind complement component C3d could better predict ABMR and graft loss in stable kidney transplant recipients (KTRs). METHODS: We included 220 stable KTRs in this study and screened them for DSAs from July 2013 to July 2016. RESULTS: Of the 220 KTRs, DSAs were detected in 24 (10.9%). The incidence of ABMR was 3.6% (8 of 220) overall, and C3d-DSA‒positive KTRs had a significantly higher incidence than SA-DSA‒positive KTRs (63.3% vs 38.9%, P = .03). Most C3d-binding DSAs were anti-HLA class II antibodies (11 of 13, 84.6%). Class II C3d-binding DSA was also significantly associated with graft failure on multivariate analysis, as were ABMR, chronic ABMR, and high serum creatinine. Class II C3d-binding DSA was also significantly associated with lower graft survival after ABMR. CONCLUSION: C3d-binding DSA, especially class II, was significantly associated with the risk of ABMR and graft loss in stable KTRs. We suggest that monitoring of stable KTRs for C3d-binding DSA, followed by biopsy, could aid in early recognition of ABMR and prevention of graft loss.

Clinical impact of complement (C1q, C3d) binding De Novo donor-specific HLA antibody in kidney transplant recipients.

INTRODUCTION: Complement binding activity of donor-specific HLA antibodies (DSA) has been suggested as a new tool to stratify immunologic risk in kidney transplantation (KT). The objective of this study was to evaluate the clinical implication of C1q/C3d binding activity of de novo DSA (dnDSA) in KT recipients. MATERIAL AND METHODS: A total of 161 pretransplant DSA-negative recipients were monitored for dnDSA at the time of biopsy. C1q/C3d binding activities of dnDSA were assessed using C1qScreen assay (One lambda, USA) and Lifecodes C3d detection assay (Immucor, USA), respectively. Clinical outcomes including biopsy-proven antibody mediated rejection (AMR), C4d detection and post-biopsy graft survival were investigated. RESULTS: De-novo DSAs were detected in fifty-four (33.5%) patients (HLA class I only, n = 19; class II only, n = 29; both class I and II, n = 6). Of them, complement binding activities were detected in 26 (48.1%) patients, including 17 C1q+ and 24 C3d+ patients. Both C1q and C3d positivity were associated with increased mean fluorescence intensity values of dnDSA. Complement binding activity of dnDSA enhanced the incidence of AMR (25.0% in C1q-C3d-, 36.4% in C1q+/C3d- or C1q-/C3d+, and 60.0% in C1q+/C3d+ patients) (P <0.001). The incidence of AMR was not different between patients with C1q+ and those with C3d+ dnDSA (64.7%, 11/17 versus 45.8%, 11/24, P = 0.238). In comparison between C1q and C3d assay according to HLA specificity, C1q+ HLA class I ± II dnDSA was the best predictor for AMR (odds ratio: 27.2). C1q+/C3d+ dnDSA was associated with more C4d deposition in allograft tissue and inferior post-biopsy graft survival. Clinical outcomes were not significantly different between C1q+ and C3d+ dnDSA-positive patients. CONCLUSION: Detection of complement binding activity using both C1q and C3d assays can be a further prognostic marker for predicting AMR and allograft outcome in dnDSA+ kidney transplant patients.

Pretransplant C3d-Fixing Donor-Specific Anti-HLA Antibodies Are Not Associated with Increased Risk for Kidney Graft Failure.

Background Complement-fixing antibodies against donor HLA are considered a contraindication for kidney transplant. A modification of the IgG single-antigen bead (SAB) assay allows detection of anti-HLA antibodies that bind C3d. Because early humoral graft rejection is considered to be complement mediated, this SAB-based technique may provide a valuable tool in the pretransplant risk stratification of kidney transplant recipients.Methods Previously, we established that pretransplant donor-specific anti-HLA antibodies (DSAs) are associated with increased risk for long-term graft failure in complement-dependent cytotoxicity crossmatch-negative transplants. In this study, we further characterized the DSA-positive serum samples using the C3d SAB assay.Results Among 567 pretransplant DSA-positive serum samples, 97 (17%) contained at least one C3d-fixing DSA, whereas 470 (83%) had non-C3d-fixing DSA. At 10 years after transplant, patients with C3d-fixing antibodies had a death-censored, covariate-adjusted graft survival of 60%, whereas patients with non-C3d-fixing DSA had a graft survival of 64% (hazard ratio, 1.02; 95% confidence interval, 0.70 to 1.48 for C3d-fixing DSA compared with non-C3d-fixing DSA; P=0.93). Patients without DSA had a 10-year graft survival of 78%.Conclusions The C3d-fixing ability of pretransplant DSA is not associated with increased risk for graft failure.

Understanding the Correlation Between DSA, Complement Activation, and Antibody-Mediated Rejection in Heart Transplant Recipients.

BACKGROUND: Donor-specific HLA antibodies (DSA) are associated with increased rates of rejection and of graft failure in cardiac transplantation. The goal of this study was to determine the association of preformed and posttransplant development of newly detected DSA (ndDSA) with antibody-mediated rejection (AMR) and characterize the clinical relevance of complement-activating DSA in heart allograft recipients. METHODS: The study included 128 adult and 48 pediatric heart transplant patients transplanted between 2010 and 2013. Routine posttransplant HLA antibody testing was performed by IgG single-antigen bead test. The C3d single-antigen bead assay was used to identify complement-activating antibodies. Rejection was diagnosed using International Society for Heart and Lung Transplantation criteria. RESULTS: In this study, 22 patients were transplanted with preexisting DSA, and 43 patients developed ndDSA posttransplant. Pretransplant (P < 0.05) and posttransplant (P < 0.001) ndDSA were associated with higher incidence of AMR. Patients with C3d + DSA had significantly higher incidence of AMR compared with patients with no DSA (P < 0.001) or patients with C3d-DSA (P = 0.02). Nine (36%) of 25 patients with AMR developed transplant coronary artery disease compared with 17 (15.9%) of 107 patients without AMR (P < 0.05). Among the 47 patients who received ventricular assistant device (VAD), 7 of 9 VAD+ patients with preformed DSA experienced AMR compared with 7 of 38 VAD+ patients without preformed DSA, indicating presensitization to donor HLA significantly increased the risk of AMR (P < 0.01). CONCLUSIONS: Preformed and posttransplant ndDSA were associated with AMR. C3d + DSA correlates with complement deposition on the graft and higher risk of AMR which may permit the application of personalized immunotherapy targeting the complement pathway.

Complement activation in acute myocardial infarction: An early marker of inflammation and tissue injury?

BACKGROUND: Acute myocardial infarction (AMI) is a potentially fatal condition, being a major cause of death worldwide. Ischemia suffered during AMI causes tissue damage, leading to an inflammatory process. Moreover, myocardial injury can generate damage-associated molecular patterns that activate pattern recognition molecules including some complement proteins. METHODS: Here we investigated products of complement activation, C3d and soluble C5b9 (sC5b9), as potential biomarkers for myocardial injury and inflammation, as well as serum cytokines (IL-6 and TNF-alpha), alpha-1-acid glycoprotein (AGP), and classical markers of myocardial necrosis (creatine kinase, creatine kinase-MB isoform, myoglobin and troponin-I) in a longitudinal study of patients with AMI (from admission, 6 h and 12 h post admission, and at discharge from hospital). Individuals undergoing cardiac catheterization (CC) with normal coronary arteries and asymptomatics with no history of cardiovascular disease or invasive procedures were included as controls. RESULTS: Plasma C3d was higher in AMI at admission, 6 h, 12 h, and discharge vs CC (p < 0.0001; p = 0.0061; p = 0.0081; p = 0.044) and asymptomatic (p = 0.0001 for admission, 6 h and 12 h; p = 0.0002 for discharge). Moreover, sC5b9 was higher only at admission and 6 h vs asymptomatic (p = 0.0031 and p = 0.0019). Additionally, AGP levels were elevated at admission, 6 h, 12 h, and discharge vs asymptomatic (p = 0.0003; p = 0.0289; p = 0.0009, p = 0.0017). IL-6 concentration was low at admission and 6 h and reached a peak at 12 h (p < 0.0001 for all groups). All classical markers of myocardial necrosis presented higher concentration at 6 h. CONCLUSIONS: Our results showed that complement activation is an early event in AMI occurring before the elevation of classical markers of myocardial necrosis such as creatine kinase, creatine kinase-MB isoform, myoglobin and troponin-I. These findings indicated C3d and sC5b9 as possible biomarkers for inflammation and tissue damage in AMI.

Clinical utility of complement-dependent C3d assay in kidney recipients presenting with late allograft dysfunction.

The objective of this study was to evaluate the utility of a complement-dependent C3d assay to risk stratify donor-specific antibodies (DSA) in a multicenter cohort of kidney recipients presenting with new-onset clinical dysfunction. A total of 106 subjects with evidence of DSA at a mean period of 5.3 ± 5.0 years posttransplant underwent testing using C3d reagents. C3d positivity was strongly associated with both the peak and sum IgG DSA MFI, with 98.3% (n = 57/58) of strongly reactive sera (peak MFI > 10 000) eliciting a positive signal. Patients with C3d+ DSA had a higher creatinine (P = .03), more significant graft fibrosis (P = .035), and a faster rate of graft loss posttest compared to those with C3d- DSA (P = .05). Subanalysis of patients with low-moderate level DSA confirmed the inferior outcome associated with C3d positivity. Despite the prognostic value of C3d as a stand-alone test, the assay did not provide independent risk prediction after incorporation of graft fibrosis in a multivariate model (P = .94). Overall, C3d offered limited discriminatory value for strong DSA with peak IgG MFI > 10 000 and in patients where histologic data is available, but its utilization may be considered in those with low-moderate level DSA and where an allograft biopsy is not accessible.

A novel C3d-containing oligomeric vaccine provides insight into the viability of testing human C3d-based vaccines in mice.

The use of C3d, the final degradation product of complement protein C3, as a "natural" adjuvant has been widely examined since the initial documentation of its immunogenicity-enhancing properties as a consequence of binding to complement receptor 2. Subsequently it was demonstrated that these effects are most evident when oligomeric, rather than when monomeric forms of C3d, are linked to various test protein antigens. In this study, we examined the feasibility of enhancing the adjuvant properties of human C3d further by utilizing C4b-binding protein (C4BP) to provide an oligomeric arrayed scaffold fused to the model antigen, tetanus toxin C fragment (TTCF). High molecular weight, C3d-containing oligomeric vaccines were successfully expressed, purified from mammalian cells and used to immunize groups of mice. Surprisingly, anti-TTCF antibody responses measured in these mice were poor. Subsequently we established by in vitro and in vivo analysis that, in the presence of mouse C3, human C3d does not interact with either mouse or even human complement receptor 2. These data confirm the requirement to develop murine versions of C3d based adjuvant compounds to test in mice or that mice would need to be developed that express both human C3 and human CR2 to allow the testing of human C3d based adjuvants in mouse in any capacity.

Immune Effect of Newcastle Disease Virus DNA Vaccine with C3d as a Molecular Adjuvant.

Newcastle disease is a serious infectious disease in the poultry industry. The commercial vaccines can only offer limited protection and some of them are expensive and need adjuvants. At present, DNA vaccines are widely used. However, the immune responses induced by DNA vaccines are too slow and low. Here, we constructed the transfer vectors with a different number of C3d as molecular adjuvants (n = 1, 2, 4, or 6), and the vectors were cloned into the optimal eukaryotic expression plasmid (pVAXI-optiF) that expressed the F gene of Newcastle disease virus (NDV), and named pVAXI-F(o)-C3d1, pVAXI -F(o)-C3d2, pVAXI-F(o)-C3d4, and pVAXI-F(o)-C3d6, respectively. Cell transfection test indicated that pVAXI-F(o)-C3d6 showed the highest expression. In vivo immunization showed that the chickens immunized with pVAXI-F(o)-C3d6 intramuscularly induced better immune responses than the chickens immunized with the other plasmids. The protective efficacy of pVAXI-F(o)-C3d6 was 80% after challenge with the highly virulent NDV strain F48E9. The results in this study showed that C3d6 could be used as a molecular adjuvant to quickly induce an effective immune response to control NDV.

Deleterious impact of C3d-binding donor-specific anti-HLA antibodies after pediatric liver transplantation.

BACKGROUND: The prevalence and clinical impact of anti-HLA donor-specific antibodies (DSA) after liver transplantation (LT) have not been extensively studied, especially in pediatric population. METHODS: The present cross-sectional study included 100 patients who underwent a first LT in childhood. Anti HLA immunization study was performed at a single time point during routine follow-up using Luminex® single antigen tests with classical anti-IgG conjugate and anti-C3d conjugate. RESULTS: The main indication for LT was biliary atresia (52%) and median age at LT was 4.6years. The median time between LT and DSA assessment was 7.8years (range 1-21years). DSA was identified in twenty-four patients (24%) after LT, with a prevalence of 8%, 28%, 33%, 50%, respectively 0-5years, 5-10years, 10-15years and >15years after LT. DSA were mainly class II (23/24) with a mean MFI of 9.731±5.489 and 18 (79.3%) were C3d-binding DSA. Multivariate analysis disclosed that time elapsed since LT (p<0.01) and history of fulminant hepatitis (p=0.04) were significantly associated with a higher rate of DSA. Liver function tests (at time of DSA assessment) were not different according to the presence or not of DSA (or C3d-binding DSA). Regarding histology, the DSA group had a higher rate of chronic rejection, cirrhosis and centrilobular fibrosis or cirrhosis. In addition, patients with C3d-binding DSA and high MFI (>10,000) had a significant poorer long-term graft survival (p=0.03). CONCLUSION: In our pediatric cohort of LT, prevalence of DSA was high and increased regularly with time. Presence of C3d positive-DSA with high MFI was associated with a higher rate of graft loss.