Serum complement proteins rather than inflammatory factors is effective in predicting psychosis in individuals at clinical high risk.

Immunological/inflammatory factors are implicated in the development of psychosis. Complement is a key driver of inflammation; however, it remains unknown which factor is better at predicting the onset of psychosis. This study aimed to compare the alteration and predictive performance of inflammation and complement in individuals at clinical high risk (CHR). We enrolled 49 individuals at CHR and 26 healthy controls (HCs). Twenty-five patients at CHR had converted to psychosis (converter) by the 3-year follow-up. Inflammatory cytokines, including interleukin (IL)-1ß, 6, 8, 10, tumor necrosis factor-alpha (TNF-alpha), macrophage colony-stimulating factor levels, and complement proteins (C1q, C2, C3, C3b, C4, C4b, C5, C5a, factor B, D, I, H) were measured by enzyme-linked immunosorbent assay at baseline. Except for TNF- alpha, none of the inflammatory cytokines reached a significant level in either the comparison of CHR individuals and HC or between CHR-converters and non-converters. The C5, C3, D, I, and H levels were significantly lower (C5, p = 0.006; C3, p = 0.009; D, p = 0.026; I, p = 0.016; H, p = 0.019) in the CHR group than in the HC group. Compared to non-converters, converters had significantly lower levels of C5 (p = 0.012) and C5a (p = 0.007). None of the inflammatory factors, but many complement factors, showed significant correlations with changes in general function and symptoms. None of the inflammatory markers, except for C5a and C5, were significant in the discrimination of conversion outcomes in CHR individuals. Our results suggest that altered complement levels in the CHR population are more associated with conversion to psychosis than inflammatory factors. Therefore, an activated complement system may precede the first-episode of psychosis and contribute to neurological pathogenesis at the CHR stage.

Complement C4A Regulates Autoreactive B Cells in Murine Lupus.

Systemic lupus erythematosus (SLE) is a severe autoimmune disease mediated by pathogenic autoantibodies. While complement protein C4 is associated with SLE, its isoforms (C4A and C4B) are not equal in their impact. Despite being 99% homologous, genetic studies identified C4A as more protective than C4B. By generating gene-edited mouse strains expressing either human C4A or C4B and crossing these with the 564lgi lupus strain, we show that, overall, C4A-like 564Igi mice develop less humoral autoimmunity than C4B-like 564Igi mice. This includes a decrease in the number of GCs, autoreactive B cells, autoantibodies, and memory B cells. The higher efficiency of C4A in inducing self-antigen clearance is associated with the follicular exclusion of autoreactive B cells. These results explain how the C4A isoform is protective in lupus and suggest C4A as a possible replacement therapy in lupus.

CD46 knock-out using CRISPR/Cas9 editing of hTERT immortalized human cells modulates complement activation.

The kidney is especially sensitive to diseases associated with overactivation of the complement system. While most of these diseases affect kidney glomeruli and the microvasculature, there is also evidence for tubulointerstitial deposition of complement factors. Complement inactivating factors on cell membranes comprise CD55, CD59 and CD46, which is also termed membrane cofactor protein (MCP). CD46 has been described as localized to glomeruli, but especially also to proximal tubular epithelial cells (RPTECs). However, human cell culture models to assess CD46 function on RPTECs are still missing. Therefore, we here performed gene editing of RPTEC/TERT1 cells generating a monoclonal CD46-/- cell line that did not show changes of the primary cell like characteristics. In addition, factor I and CD46-mediated cleavage of C4b into soluble C4c and membrane deposited C4d was clearly reduced in the knock-out cell line as compared to the maternal cells. Thus, human CD46-/- proximal tubular epithelial cells will be of interest to dissect the roles of the epithelium and the kidney in various complement activation mediated tubulointerstitial pathologies or in studying CD46 mediated uropathogenic internalization of bacteria. In addition, this gives proof-of-principle, that telomerized cells can be used in the generation of knock-out, knock-in or any kind of reporter cell lines without losing the primary cell characteristics of the maternal cells.

The structural basis for inhibition of the classical and lectin complement pathways by S. aureus extracellular adherence protein.

The extracellular adherence protein (Eap) plays a crucial role in pathogenesis and survival of Staphylococcus aureus by inhibiting the classical and lectin pathways of complement. We have previously shown that Eap binds with nanomolar affinity to complement C4b and disrupts the initial interaction between C4b and C2, thereby inhibiting formation of the classical and lectin pathway C3 pro-convertase. Although an underlying mechanism has been identified, the structural basis for Eap binding to C4b is poorly understood. Here, we show that Eap domains 3 and 4 each contain a low-affinity, but saturable binding site for C4b. Taking advantage of the high lysine content of Eap, we used a zero-length crosslinking approach to map the Eap binding site to both the α'- and γ-chains of C4b. We also probed the C4b/Eap interface through a chemical footprinting approach involving lysine modification, proteolytic digestion, and mass spectrometry. This identified seven lysines in Eap that undergo changes in solvent exposure upon C4b binding. We found that simultaneous mutation of these lysines to either alanine or glutamate diminished C4b binding and complement inhibition by Eap. Together, our results provide insight into Eap recognition of C4b, and suggest that the repeating domains that comprise Eap are capable of multiple ligand-binding modes.

Domain structure of human complement C4b extends with increasing NaCl concentration: implications for its regulatory mechanism.

During the activation of complement C4 to C4b, the exposure of its thioester domain (TED) is crucial for the attachment of C4b to activator surfaces. In the C4b crystal structure, TED forms an Arg104-Glu1032 salt bridge to tether its neighbouring macroglobulin (MG1) domain. Here, we examined the C4b domain structure to test whether this salt bridge affects its conformation. Dual polarisation interferometry of C4b immobilised at a sensor surface showed that the maximum thickness of C4b increased by 0.46 nm with an increase in NaCl concentration from 50 to 175 mM NaCl. Analytical ultracentrifugation showed that the sedimentation coefficient s20,w of monomeric C4b of 8.41 S in 50 mM NaCl buffer decreased to 7.98 S in 137 mM NaCl buffer, indicating that C4b became more extended. Small angle X-ray scattering reported similar RG values of 4.89-4.90 nm for C4b in 137-250 mM NaCl. Atomistic scattering modelling of the C4b conformation showed that TED and the MG1 domain were separated by 4.7 nm in 137-250 mM NaCl and this is greater than that of 4.0 nm in the C4b crystal structure. Our data reveal that in low NaCl concentrations, both at surfaces and in solution, C4b forms compact TED-MG1 structures. In solution, physiologically relevant NaCl concentrations lead to the separation of the TED and MG1 domain, making C4b less capable of binding to its complement regulators. These conformational changes are similar to those seen previously for complement C3b, confirming the importance of this salt bridge for regulating both C4b and C3b.

C4d as a Diagnostic Tool in Proliferative GN.

Proliferative GN is classified as immune complex-mediated or complement-mediated (C3 glomerulopathy). Immune complex-mediated GN results from glomerular deposition of immune-complexes/Ig and C3; the C3 is derived from activation of the classical and/or lectin pathways of complement. C3 glomerulopathy results from deposition of C3 and other complement fragments with minimal or no deposition of immune complexes/Ig; the C3 is derived from activation of the alternative pathway of complement. C4d is a byproduct of activation of the classic and lectin pathways. Although widely used as a marker for antibody-mediated rejection, the significance of C4d in C3 glomerulopathy is undetermined. We studied glomerular C4d staining in 18 biopsy specimens of immune-complex GN, 30 biopsy specimens of C3 GN, and 13 biopsy specimens of postinfectious GN. All specimens of immune complex-mediated GN, except two specimens of IgA nephropathy and one specimen of sclerosing membranoproliferative GN, showed bright (2-3+) C4d staining. The staining pattern of C4d mirrored the staining patterns of Ig and C3. Conversely, C4d staining was completely negative in 24 (80%) of 30 specimens of C3 glomerulopathy, and only trace/1+ C4d staining was detected in six (20%) specimens. With regard to postinfectious GN, C4d staining was negative in six (46%) of 13 specimens, suggesting an abnormality in the alternative pathway, and it was positive in seven (54%) specimens. To summarize, C4d serves as a positive marker for immune complex-mediated GN but is absent or minimally detected in C3 glomerulopathy.

Structural Basis for the Function of Complement Component C4 within the Classical and Lectin Pathways of Complement.

Complement component C4 is a central protein in the classical and lectin pathways within the complement system. During activation of complement, its major fragment C4b becomes covalently attached to the surface of pathogens and altered self-tissue, where it acts as an opsonin marking the surface for removal. Moreover, C4b provides a platform for assembly of the proteolytically active convertases that mediate downstream complement activation by cleavage of C3 and C5. In this article, we present the crystal and solution structures of the 195-kDa C4b. Our results provide the molecular details of the rearrangement accompanying C4 cleavage and suggest intramolecular flexibility of C4b. The conformations of C4b and its paralogue C3b are shown to be remarkably conserved, suggesting that the convertases from the classical and alternative pathways are likely to share their overall architecture and mode of substrate recognition. We propose an overall molecular model for the classical pathway C5 convertase in complex with C5, suggesting that C3b increases the affinity for the substrate by inducing conformational changes in C4b rather than a direct interaction with C5. C4b-specific features revealed by our structural studies are probably involved in the assembly of the classical pathway C3/C5 convertases and C4b binding to regulators.

Acute Rejection Clinically Defined Phenotypes Correlate With Long-term Renal Allograft Survival.

BACKGROUND: Classification of acute rejection (AR) based on etiology and timing may provide a means for enhancing therapeutic results and allograft survival. This study evaluated graft and patient survival after the first AR episodes among kidney transplant recipients with an early or late antibody-mediated rejection (AMR), acute cellular rejection (ACR) or mixed AR (MAR). METHODS: A prospective institutional review board-approved database was queried to identify biopsy-proven first AR episodes occurring from January 2005 to October 2012. The ACR was defined by Banff criteria; borderline AR was excluded. The AMR was defined as 3 of 4 criteria: renal dysfunction, donor specific antibody, C4d positivity on biopsy, and histological changes. The MAR met criteria for both ACR and AMR. Early AR occurred within six months post-transplant. AR episodes were then assigned to 1 of the 6 categories--early AMR, early ACR, early MAR, late AMR, late ACR, and late MAR. RESULTS: One hundred eighty-two kidney transplant recipients identified with a first AR episode. Mean follow-up was 773 days (± 715 days). No difference was observed in patient survival. Death-censored graft survival was 84%. Death-censored graft loss was higher with late versus early AMR (P = 0.01) and late versus early ACR (P = 0.03), but not late versus early MAR (P = 0.3). CONCLUSIONS: The AR type demonstrated a hierarchy for graft survival with ACR better than MAR better than AMR, which persisted for both early and late AR. Improvement in long-term results of AR may require development of specific treatment for individual AR types.

Antibodies reactive to cleaved sites in complement proteins enable highly specific measurement of soluble markers of complement activation.

An emerging number of diseases and therapeutic approaches with defined involvement of the complement system justify a need for specific markers reflecting activation of particular effector arms of the complement cascade. Measurement of such soluble markers in circulation is a challenge since the specificity of antibodies must be limited to activated complement fragments but not predominant and ubiquitous parental molecules. Existing assays for the measurement of soluble, activated complement proteins are based on the detection of conformational neoepitopes. We tested an alternative approach based on detection of short linear neoepitopes exposed at the cleavage sites after activation of the actual complement component. Obtained antibodies reactive to C4d and C5b fragments enabled us to set up highly specific sandwich ELISAs, which ensured trustful measurements without false positive readouts characteristic for some of the widely used assays.

Hematopoietic stem cell transplantation-associated thrombotic microangiopathy accompanied by renal arteriolar C4d deposition.

HSCT-associated thrombotic microangiopathy (TA-TMA) is a severe complication with a poor prognosis. Recently, it has been reported that complement system dysregulation, such as CFH autoantibodies and deletions CFH-related genes 3 and 1, induced TA-TMA. In addition, C4d-positive renal arterioles are both a good marker of complement system activation and a useful diagnostic tool for TA-TMA. Because dysregulation of the complement system is associated with TA-TMA, the complement system might be a therapeutic target, such as eculizumab, a terminal complement inhibitor. Herein, we describe an eight-yr-old boy who developed TA-TMA accompanied by severe renal dysfunction. His renal specimen showed diffuse C4d deposition in the renal arterioles, which is consistent with TA-TMA. Although the patient gradually improved without eculizumab, renal arteriolar C4d staining in sample with TA-TMA shows the complement system activation and may guide the target therapy using the eculizumab.

Time-dependent specificity of immunopathologic (C4d-CD68) and histologic criteria of antibody-mediated rejection for donor-specific antibodies and allograft dysfunction in heart transplantation.

BACKGROUND: In heart transplantation, antibody-mediated rejection (AMR) is diagnosed and graded on the basis of immunopathologic (C4d-CD68) and histopathologic criteria found on endomyocardial biopsies (EMB). Because some pathologic AMR (pAMR) grades may be associated with clinical AMR, and because humoral responses may be affected by the intensity of immunosuppression during the first posttransplantation year, we investigated the incidence and positive predictive values (PPV) of C4d-CD68 and pAMR grades for clinical AMR as a function of time. METHODS: All 564 EMB from 40 adult heart recipients were graded for pAMR during the first posttransplantation year. Clinical AMR was diagnosed by simultaneous occurrence of pAMR on EMB, donor specific antibodies and allograft dysfunction. RESULTS: One patient demonstrated clinical AMR at postoperative day 7 and one at 6 months (1-year incidence 5%). C4d-CD68 was found on 4,7% EMB with a "decrescendo" pattern over time (7% during the first 4 months vs. 1.2% during the last 8 months; P < 0.05). Histopathologic criteria of AMR occurred on 10.3% EMB with no particular time pattern. Only the infrequent (1.4%) pAMR2 grade (simultaneous histopathologic and immunopathologic markers) was predictive for clinical AMR, particularly after the initial postoperative period (first 4 months and last 8 months PPV = 33%-100%; P < 0.05). CONCLUSION: In the first posttransplantation year, AMR immunopathologic and histopathologic markers were relatively frequent, but only their simultaneous occurrence (pAMR2) was predictive of clinical AMR. Furthermore, posttransplantation time may modulate the occurrence of C4d-CD68 on EMB and thus the incidence of pAMR2 and its relevance to the diagnosis of clinical AMR.

Deciphering complement interference in anti-human leukocyte antigen antibody detection with flow beads assays.

BACKGROUND: Anti-human leukocyte antigen (HLA) antibody detection in solid-phase flow beads assays can be quenched by complement activation, but the precise mechanism of this interference is not fully elucidated yet. METHODS: Using the Luminex flow beads screening assay for detection of anti-HLA antibodies, we analyzed the binding of high concentrations of the pan class I anti-HLA monoclonal antibody W6/32 in neat normal, ethylenediaminetetraacetic acid-treated normal and complement factors C1q, C4/C3, C2, C3, factor B or C5-depleted human sera, using anti-mouse immunoglobulin G as the detection antibody. Complement activation and binding to beads were revealed using anti-human C1q, C4d, and C3d antibodies. To translate our findings to the human setting, we used the class I and class II HLA single-antigen flow beads assays and sera from four patients with high titers of antibodies. RESULTS: Detection of W6/32 did not suffer any interference with C1q and C4/C3-depleted sera. A partial quenching was observed with C2, C3, and factor B-depleted sera, but was more pronounced with the factor B-depleted serum. W6/32 was undetectable in presence of C5-depleted serum. The binding of activation products derived from C3 principally, and also from C4, impaired immunoglobulin G and C1q detection. Accordingly, C4d detection was hindered by deposition of activated C3. Similar findings were obtained with patients' sera. CONCLUSION: Binding of C4 and C3 activation products is the main responsible for complement interference in flow beads assays. A complete quenching requires complement activation through C3 cleavage and its amplification by the alternative pathway.

The severity of acute cellular rejection defined by Banff classification is associated with kidney allograft outcomes.

BACKGROUND: It is unclear if the severity or the timing of acute cellular rejection (ACR) defined by Banff classification 2009 is associated with graft survival. METHODS: Borderline changes, TCMR I (interstitial rejection), and TCMR II/III (vascular rejection) were defined as low, moderate, and high ACR severity, respectively. Approximately 270 patients who had at least one episode of ACR were enrolled, 270 biopsies were chosen which showed the highest ACR severity of each patient and were negative for donor-specific antibodies (DSA), C4d, and microcirculation changes (MC). Six months were used as the cutoff to define early and late ACR; 370 patients without biopsy posttransplantation were recruited in the control group. RESULTS: Up to 8-year posttransplantation, death-censored graft survival (DCGS) rates of control, borderline, TCMR I, and TCMR II/III groups were 97.6%, 93.3%, 79.6%, and 73.6% (log rank test, P<0.001); the control group had significantly higher DCGS rate than the three ACR groups (each pairwise comparison yields P<0.05). The DCGS rate of late ACR was significantly lower compared with early ACR (63.6% vs. 87.4%, P<0.001). Intimal arteritis (Banff v-lesion) was an independent histologic risk factor correlated with long-term graft loss regardless of the timing of ACR. The v-lesions with minimal or high-grade tubulitis displayed similar graft survival (72.7% vs. 72.9%, P=0.96). CONCLUSION: All types of ACR affect long-term graft survival. Vascular or late ACR predict poorer graft survival; the extent of tubulointerstitial inflammation (TI) is of no prognostic significance for vascular rejection.

Yersinia pestis Ail recruitment of C4b-binding protein leads to factor I-mediated inactivation of covalently and noncovalently bound C4b.

The outer membrane protein Ail of Yersinia pestis mediates several virulence functions, including serum resistance. Here, we demonstrate that Ail binds C4b-binding protein (C4BP), the primary fluid-phase regulator of the classical and lectin pathways. Noncovalent binding of C4 and C4b to Ail was also observed. C4BP bound to Ail can act as a cofactor to the serine protease factor I (fI) in the cleavage of fluid-phase C4b. Employing a panel of C4BP alpha-chain mutants, we observed that the absence of complement control protein domain 6 and 8 reduced binding to Ail. Immunoblot analysis of normal human serum (NHS)-treated bacteria revealed minimal C4b alpha'-chain complexes with bacterial outer membrane targets. Addition of the anti-C4BP monoclonal antibody MK104 to NHS restored C4b-alpha' chain target complexes, suggesting that C4b binds covalently to targets on the Y. pestis surface. C4b bound to Ail noncovalently was also cleaved in a C4BP and fI-dependent manner, leaving the C4c fragment bound to Ail. MK104 also prevented the cleavage of noncovalently bound C4b. Collectively, these data suggest that when C4BP is bound to Ail, fI can cleave and inactivate C4b that has bound covalently to bacterial surface structures as well as C4b bound noncovalently to Ail.

Using mutagenesis and structural biology to map the binding site for the Plasmodium falciparum merozoite protein PfRh4 on the human immune adherence receptor.

To survive and replicate within the human host, malaria parasites must invade erythrocytes. Invasion can be mediated by the P. falciparum reticulocyte-binding homologue protein 4 (PfRh4) on the merozoite surface interacting with complement receptor type 1 (CR1, CD35) on the erythrocyte membrane. The PfRh4 attachment site lies within the three N-terminal complement control protein modules (CCPs 1-3) of CR1, which intriguingly also accommodate binding and regulatory sites for the key complement activation-specific proteolytic products, C3b and C4b. One of these regulatory activities is decay-accelerating activity. Although PfRh4 does not impact C3b/C4b binding, it does inhibit this convertase disassociating capability. Here, we have employed ELISA, co-immunoprecipitation, and surface plasmon resonance to demonstrate that CCP 1 contains all the critical residues for PfRh4 interaction. We fine mapped by homologous substitution mutagenesis the PfRh4-binding site on CCP 1 and visualized it with a solution structure of CCPs 1-3 derived by NMR and small angle x-ray scattering. We cross-validated these results by creating an artificial PfRh4-binding site through substitution of putative PfRh4-interacting residues from CCP 1 into their homologous positions within CCP 8; strikingly, this engineered binding site had an ∼30-fold higher affinity for PfRh4 than the native one in CCP 1. These experiments define a candidate site on CR1 by which P. falciparum merozoites gain access to human erythrocytes in a non-sialic acid-dependent pathway of merozoite invasion.

Suppression of Th1 cytokine production by a peptide derived from C4b.

OBJECTIVES: The complement system has been proposed to play a significant role in the regulation of T-cell responses. However, the precise mechanism underlying C4-induced immune tolerance remains to be clarified. We recently reported that monomeric C4b inhibits CXCL10 production from blood cells. The purpose of this study was to verify the active site of monomeric C4b. MATERIALS AND METHODS: We investigated the in vitro effects of a C4b-derived peptide (VPAGSARPVAFSVVPTAAA), named HP2 (highly homologous peptide 2), on the IFN-ß-induced production of CXCL10 in human blood and the in vivo effects of the administration of HP2 on Th1/2 cytokine production in the spleen in mice. We also tested whether the administration of HP2 influences symptoms of experimentally induced ulcerative colitis in mice. RESULTS: HP2 inhibited CXCL10 production in human blood, and the administration of HP2 significantly suppressed the production of Th1 cytokines, such as IL-2, IFN-γ, and TNF-α, in spleen cells isolated from mice. The administration of HP2 in the mice significantly improved the symptoms of colitis, with down-regulation of colitogenic CD4(+)CD45RB(high) T cells and up-regulation of CD4(+)LAP/TGF-ß1(+) T cells. CONCLUSION: The amino acid sequence described above is suggested to be the active site in C4b for the inhibition of Th1 cytokine production. These results should contribute to the development of new drugs suppressing autoimmune responses.

Failure of bortezomib to cure acute antibody-mediated rejection in a non-compliant renal transplant patient.

Bortezomib has appeared recently as a potential active treatment for acute AMR for few years. We reported a patient who received two courses of bortezomib for the treatment of an acute AMR associated with de novo HLA DSA that occurred 18 months after renal transplantation because of non-compliance. Graft biopsy revealed features of acute humoral rejection with plasmocyte infiltration and C4d staining. Bortezomib was associated with corticosteroid pulses, IVIgs, and PP. Despite this rapid management, the patient lost his graft and carried on dialysis. Bortezomib therapy in addition to current therapy of AMR is not always effective in the treatment for late acute AMR in renal transplantation. We discuss on the place of such a treatment and other therapeutic strategies in this indication.

Pretransplant donor-specific anti-HLA antibodies as predictors of early allograft rejection in ABO-compatible liver transplantation.

The significance of preexisting donor-specific HLA antibodies (HLA-DSAs) for liver allograft function is unclear. Our previous studies have shown that humoral alloreactivity frequently accompanies acute cellular rejection (ACR). In the present study, we set out to determine whether pretransplant HLA-DSAs correlate with clinically significant ACR in the first 90 days after transplantation and, if so, to determine their predictive values. Class I HLA-DSAs and class II HLA-DSAs were determined by single-antigen bead flow cytometry for 113 consecutive adult transplants. A statistical analysis was performed for data from 109 consecutive patients with graft survival greater than or equal to 90 days. All patients who developed biochemical graft dysfunction underwent liver biopsy for hematoxylin-eosin and complement component 4d staining. Cox proportional hazards models and associated hazard ratios revealed a significant association of pretransplant HLA-DSAs with clinically significant ACR: this association started with a mean fluorescence intensity (MFI) as low as 300 for both class I (hazard ratio = 2.7, P < 0.01) and class II (hazard ratio = 6.0, P < 0.01). Pretransplant HLA-DSAs were associated with an increased risk of ACR: P < 0.01 for class I (42% versus 18%), P < 0.001 for class II (37% versus 7%), and P < 0.001 for either class I or II (36% versus 3%). Class I or II HLA-DSAs with an MFI ≥ 1000 had the best positive predictive value for clinically significant ACR at 46%, whereas class I or II HLA-DSAs with an MFI ≥ 300 had the best negative predictive value at 97.1%. Although our study was based on consecutive patients, it was limited by the relatively low number of single-center subjects. In conclusion, the present study indicates that pretransplant HLA-DSAs, even at low levels of allosensitization, correlate with the risk of clinically significant ACR. Our findings suggest that anti-human leukocyte antigen antibodies could serve as donor-specific markers of immunoreactivity to the liver graft.