Diagnostic and prognostic value of mannan-binding lectin associated protein (MAp19) for functionally relevant coronary artery disease.

BACKGROUND: This study aimed to evaluate the diagnostic and prognostic potential of MAp19, a regulating component of the lectin pathway of the complement system, in patients with suspected functionally relevant coronary artery disease (fCAD) as well as the determinants of MAp19 levels. METHODS: The presence of fCAD was adjudicated using myocardial perfusion imaging with single-photon emission tomography and, where available, coronary angiography. MAp19 levels were measured in participants at rest, at peak stress tests, and two hours after the stress. The study also tracked major cardiovascular events, including non-fatal myocardial infarction and cardiovascular death, over a five-year follow-up period. RESULTS: Among the 1,571 patients analyzed (32.3 % women), fCAD was identified in 462 individuals (29.4 %). MAp19 demonstrated no diagnostic significance, yielding an area under the curve (AUC) of 0.51 (0.47-0.55). Throughout the five-year follow-up, 107 patients (6.8 %) experienced non-fatal myocardial infarctions, 99 (6.3 %) had cardiovascular death, 194 (12.3 %) experienced all cause death and 50 (3.1 %) suffered a stroke. Cox and Kaplan-Meier analysis confirmed prognostic value of MAp19 for myocardial infarction, but not for cardiovascular death. Significant increases in the concentration of MAp19 were observed during bicycle (p = 0.001) and combined stress tests (p = 0.001). CONCLUSION: MAp19 demonstrated an association with the risk of myocardial infarction. Increases in MAp19 concentration were observed during bicycle and combined stress-tests.

Inter-Alpha-Trypsin Inhibitor Heavy Chain 4 (ITIH4) as a Compensatory Protease Inhibitor in Hereditary Angioedema (HAE).

BACKGROUND: Hereditary angioedema (HAE) is a genetic disorder that manifests as recurrent angioedema attacks, most frequently due to absent or reduced C1 inhibitor (C1-INH) activity. C1-INH is a crucial regulator of enzymatic cascades in the complement, fibrinolytic, and contact systems. Inter-alpha-trypsin inhibitor heavy chain 4 (ITIH4) is an abundant plasma protease inhibitor that can inhibit enzymes in the proteolytic pathways associated with HAE. Nothing is known about its role in HAE. OBJECTIVE: Investigate ITIH4 activation in HAE, establish it as a potential biomarker, and explore its involvement in HAE-associated proteolytic pathways. METHODS: Specific immunoassays for non-cleaved ITIH4 (Intact ITIH4) and an assay detecting both intact and cleaved ITIH4 (Total ITIH4) were developed. We initially tested serum samples from HAE patients (n=20), ACEI-induced edema patients (ACEI) (n=20), and unknown HAE patients (U-HAE) (n=20). Validation involved an extended cohort of 80 HAE patients (60 type I, 20 type II), including samples taken at attack and quiescent disease periods, as well as 100 healthy controls. RESULTS: In 63% of HAE patients, the Intact ITIH4 assay showed lower signals than the Total ITIH4 assay. This difference was not observed in ACEI and U-HAE patients. Western blotting confirmed cleaved ITIH4 in low-Intact ITIH4 samples. In serum samples lacking intact endogenous ITIH4, we observed immediate cleavage of added recombinant ITIH4 suggesting continuous enzymatic activity in the serum. Confirmatory HAE cohort analysis revealed significantly lower intact ITIH4 levels in both HAE type 1 and type 2 patients compared to controls, with consistently low Intact/Total ITIH4 ratios during clinical HAE attacks. CONCLUSION: The disease-specific low intact ITIH4 levels highlight its unique nature in HAE. The results suggest that ITIH4 may exhibit compensatory mechanisms in HAE, suggesting its utility as a diagnostic and prognostic biomarker. The variations during quiescent and active disease periods raise intriguing questions about the dynamics of proteolytic pathways in HAE.

Effect of dapagliflozin on collectins and complement activation in plasma from patients with type 2 diabetes and albuminuria: Data from the DapKid cohort.

BACKGROUND: Sodium-glucose cotransporter 2 (SGLT- 2) inhibitors exert cardiovascular and kidney-protective effects in people with diabetes. Attenuation of inflammation could be important for systemic protection. The lectin pathway of complement system activation is linked to diabetic nephropathy. We hypothesized that SGLT-2 inhibitors lower the circulating level of pattern-recognition molecules of the lectin cascade and attenuate systemic complement activation. METHODS: Analysis of paired plasma samples from the DapKid crossover intervention study where patients with type 2 diabetes mellitus (T2DM) and albuminuria were treated with dapagliflozin and placebo for 12 weeks (10 mg/day, n=36). ELISA was used to determine concentrations of collectin kidney 1 (CL-K1), collectin liver 1 (CL-L1), mannose-binding lectin (MBL), MBL-associated serine protease 2 (MASP-2), the anaphylatoxin complement factor 3a (C3a), the stable C3 split product C3dg and the membrane attack complex (sC5b-9). RESULTS: As published before, dapagliflozin treatment lowered Hba1C from 74 (14.9) mmol/mol to 66 (13.9) mmol/mol (p<0.0001), and the urine albumin/creatinine ratio from 167.8 mg/g to 122.5 mg/g (p<0.0001). Plasma concentrations of CL-K1, CL-L1, MBL, and MASP-2 did not change significantly after dapagliflozin treatment (P>0.05) compared to placebo treatment. The plasma levels of C3a (P<0.05) and C3dg (P<0.01) increased slightly but significantly, 0.6 [0.2] units/mL and 76 [52] units/mL respectively, after dapagliflozin treatment. The C9-associated neoepitope in C5b-9 did not change in plasma concentration by dapagliflozin (P>0.05). CONCLUSION: In patients with type 2 diabetes and albuminuria, SGLT-2 inhibition resulted in modest C3 activation in plasma, likely not driven by primary changes in circulating collectins and not resulting in changes in membrane attack complex. Based on systemic analyses, organ-specific local protective effects of gliflozins against complement activation cannot be excluded.

Trypanosoma brucei Invariant Surface Glycoprotein 75 Is an Immunoglobulin Fc Receptor Inhibiting Complement Activation and Antibody-Mediated Cellular Phagocytosis.

Various subspecies of the unicellular parasite Trypanosoma brucei cause sleeping sickness, a neglected tropical disease affecting millions of individuals and domestic animals. Immune evasion mechanisms play a pivotal role in parasite survival within the host and enable the parasite to establish a chronic infection. In particular, the rapid switching of variant surface glycoproteins covering a large proportion of the parasite's surface enables the parasite to avoid clearance by the adaptive immune system of the host. In this article, we present the crystal structure and discover an immune-evasive function of the extracellular region of the T. brucei invariant surface gp75 (ISG75). Structural analysis determined that the ISG75 ectodomain is organized as a globular head domain and a long slender coiled-coil domain. Subsequent ligand screening and binding analysis determined that the head domain of ISG75 confers interaction with the Fc region of all subclasses of human IgG. Importantly, the ISG75-IgG interaction strongly inhibits both activation of the classical complement pathway and Ab-dependent cellular phagocytosis by competing with C1q and host cell FcγR CD32. Our data reveal a novel immune evasion mechanism of T. brucei, with ISG75 able to inactivate the activities of Abs recognizing the parasite surface proteins.

Diagnostic and prognostic value of H-ficolin for functionally relevant coronary artery disease.

BACKGROUND: We aimed to test the diagnostic and prognostic ability of H-ficolin, an initiator of the lectin pathway of the complement system, for functionally relevant coronary artery disease (fCAD), and explore its determinants. METHODS: The presence of fCAD was adjudicated using myocardial perfusion imaging single-photon emission tomography and coronary angiography. H-ficolin levels were measured by a sandwich-type immunoassay at rest, peak stress-test, and 2 h after stress-test. Cardiovascular death and non-fatal myocardial infarction were assessed during 5-year follow-up. RESULTS: Among 1,571 patients (32.3 % women), fCAD was detected in 462 patients (29.4 %). H-ficolin concentration at rest was 18.6 (15.3-21.8) µg/ml in patients with fCAD versus 17.8 (15.4-21.5) µg/ml, p = 0.33, in patients without fCAD, resulting in an AUC of 0.53 (95 %CI 0.48-0.56). During follow-up, 107 patients (6.8 %) had non-fatal myocardial infarction and 99 patients (6.3 %) experienced cardiovascular death. In Cox regression analysis, H-ficolin was not a predictor of events in the overall cohort. Subgroup analysis suggested a potential link between H-ficolin and non-fatal myocardial infarction in patients without fCAD (adjusted HR 1.03, 95 % CI 1.02-1.15, p = 0.005). H-ficolin concentration showed a weak positive correlation with systolic (r = 0.069, p < 0.001) and diastolic blood pressure (r = 0.111, p < 0.001). CONCLUSION: H-ficolin concentration did not have diagnostic and/or prognostic value in patients referred for fCAD work-up.

Complement Proteins L-Ficolin and M-Ficolin Are Increased in Patients With Axial Spondyloarthritis and Decrease After Tumor Necrosis Factor Inhibitor Treatment.

OBJECTIVE: We have previously reported elevated levels of the complement lectin pathway proteins L-ficolin and H-ficolin in patients with axial spondyloarthritis (axSpA) compared with healthy controls. The aim of the present study was to investigate these biomarkers in a cross-sectional cohort of patients suffering from low back pain (LBP). Further, we aimed to investigate changes in lectin pathway protein levels after initiation of adalimumab (ADA; a tumor necrosis factor inhibitor) in a longitudinal cohort of patients with axSpA. METHODS: Lectin pathway protein levels (mannan-binding lectin [MBL], collectin liver 1, H-ficolin, L-ficolin, M-ficolin, MBL-associated serine protease [MASP]-1, MASP-2, MASP-3, MBL-associated protein 19 [MAp19], and MAp44) in EDTA plasma were determined in 2 well-characterized cohorts: (1) a clinical cross-sectional cohort of patients with LBP, including patients with axSpA (n = 23), patients with unspecific LBP (uLBP) with ≥ 1 SpA features (n = 55), and patients with uLBP without SpA features or magnetic resonance imaging findings suggestive of axSpA (n = 64); and (2) a randomized double-blinded, placebo-controlled trial cohort of patients with axSpA (n = 49) initiating ADA therapy. Lectin pathway protein levels were determined using immunoassays. RESULTS: Plasma levels of L-ficolin and M-ficolin were significantly increased in the cross-sectional cohort of newly diagnosed patients with axSpA compared with clinically relevant controls with uLBP (all P < 0.05). Both L-ficolin and M-ficolin decreased significantly after ADA therapy (P < 0.05). CONCLUSION: L-ficolin and M-ficolin levels are elevated in newly diagnosed patients with axSpA compared with clinically relevant controls. Both L-ficolin and M-ficolin levels decrease significantly after initiating ADA therapy. These findings provide new insights into the inflammatory processes in axSpA and support the involvement of complement in axSpA pathogenesis.

An integrated organoid omics map extends modeling potential of kidney disease.

Kidney organoids are a promising model to study kidney disease, but their use is constrained by limited knowledge of their functional protein expression profile. Here, we define the organoid proteome and transcriptome trajectories over culture duration and upon exposure to TNFα, a cytokine stressor. Older organoids increase deposition of extracellular matrix but decrease expression of glomerular proteins. Single cell transcriptome integration reveals that most proteome changes localize to podocytes, tubular and stromal cells. TNFα treatment of organoids results in 322 differentially expressed proteins, including cytokines and complement components. Transcript expression of these 322 proteins is significantly higher in individuals with poorer clinical outcomes in proteinuric kidney disease. Key TNFα-associated protein (C3 and VCAM1) expression is increased in both human tubular and organoid kidney cell populations, highlighting the potential for organoids to advance biomarker development. By integrating kidney organoid omic layers, incorporating a disease-relevant cytokine stressor and comparing with human data, we provide crucial evidence for the functional relevance of the kidney organoid model to human kidney disease.

Differentiating between activation via the lectin or the classical complement pathway in patients with systemic lupus erythematosus.

Complement activation is a hallmark of systemic lupus erythematosus (SLE) and can proceed through the classical (CP), lectin (LP), or alternative pathway (AP). When managing SLE patients, pathway-specific complement activation is rarely monitored as clinical assays are unavailable. In this study, we aim to differentiate between CP- or LP-mediated complement activation in SLE patients by quantifying pathway-specific protein complexes, namely C1s/C1-inhibitor (C1-INH) (CP-specific activation) and MASP-1/C1-INH (LP-specific activation). Levels for both complexes were assessed in 156 SLE patients and 50 controls using two newly developed ELISAs. We investigated whether pathway-specific complement activation was associated with disease activity and lupus nephritis (LN). Disease activity stratification was performed using SLEDAI scores assessed at inclusion. C1s/C1-INH concentrations were significantly increased in active SLE patients (SLEDAI ≥6) when compared with SLE patients with low disease activity (SLEDAI <6, P < 0.01) and correlated with SLEDAI score (r = .29, P < 0.01). In active LN, MASP-1/C1-INH plasma concentrations were significantly increased compared with nonactive LN (P = 0.02). No differences in MASP-1/C1-INH plasma concentrations were observed between active SLE patients and patients with low disease activity (P = 0.11) nor did we observe a significant correlation with disease activity (r = 0.12, P = 0.15). Our data suggest that the CP and the LP are activated in SLE. The CP is activated in active SLE disease, whereas activation of the LP might be more specific to disease manifestations like LN. Our results warrant further research into specific complement pathway activation in SLE patients to potentially improve specific-targeted and tailored-treatment approaches.

Trypanosoma brucei Invariant Surface gp65 Inhibits the Alternative Pathway of Complement by Accelerating C3b Degradation.

Trypanosomes are known to activate the complement system on their surface, but they control the cascade in a manner such that the cascade does not progress into the terminal pathway. It was recently reported that the invariant surface glycoprotein ISG65 from Trypanosoma brucei interacts reversibly with complement C3 and its degradation products, but the molecular mechanism by which ISG65 interferes with complement activation remains unknown. In this study, we show that ISG65 does not interfere directly with the assembly or activity of the two C3 convertases. However, ISG65 acts as a potent inhibitor of C3 deposition through the alternative pathway in human and murine serum. Degradation assays demonstrate that ISG65 stimulates the C3b to iC3b converting activity of complement factor I in the presence of the cofactors factor H or complement receptor 1. A structure-based model suggests that ISG65 promotes a C3b conformation susceptible to degradation or directly bridges factor I and C3b without contact with the cofactor. In addition, ISG65 is observed to form a stable ternary complex with the ligand binding domain of complement receptor 3 and iC3b. Our data suggest that ISG65 supports trypanosome complement evasion by accelerating the conversion of C3b to iC3b through a unique mechanism.

Platelets and the Lectin Pathway of Complement Activation in Patients with Systemic Lupus Erythematosus or Antiphospholipid Syndrome.

Background Patients with systemic lupus erythematosus (SLE) have an increased risk of thrombosis even when they do not have antiphospholipid syndrome (APS). Interactions between complement activation and activated platelets have been suggested in SLE and APS and could play a role in the increased thrombosis risk. Objectives To explore factors potentially related to the prothrombotic pathophysiology in patients with SLE, primary APS, and healthy controls, by investigating lectin pathway proteins (LPPs), complement activation, platelet aggregation, and platelet activation. Methods This cross-sectional cohort study included 20 SLE patients, 17 primary APS, and 39 healthy controls. Flow cytometry and light transmission aggregometry were used to assess platelet activation and aggregation. Using time-resolved immunofluorometric assays, the plasma concentrations of 11 LPPs and C3dg, reflecting complement activation, were measured. Results H-ficolin plasma concentrations were higher in SLE and APS patients than in controls ( p = 0.01 and p = 0.03). M-ficolin was lower in SLE than in APS ( p = 0.01) and controls ( p = 0.03). MAp19 was higher in APS patients than in SLE patients ( p = 0.01) and controls ( p < 0.001). In APS patients, MASP-2 and C3dg correlated negatively with platelet activation. Platelet-bound fibrinogen after agonist stimulation and C3dg concentrations correlated negatively with platelet activation. Conclusion We observed significant differences between SLE and APS patients regarding complement proteins and platelet activation. Particularly the negative correlations between MASP-2 and C3dg with platelet activation only observed in APS patients suggest that interactions between complement activation and platelets differ in SLE and APS.

Bispecific Complement Engagers for Targeted Complement Activation.

Activation of the complement system represents an important effector mechanism of endogenous and therapeutic Abs. However, efficient complement activation is restricted to a subset of Abs due to the requirement of multivalent interactions between the Ab Fc regions and the C1 complex. In the present study, we demonstrate that Fc-independent recruitment of C1 by modular bispecific single-domain Abs that simultaneously bind C1q and a surface Ag can potently activate the complement system. Using Ags from hematological and solid tumors, we show that these bispecific Abs are cytotoxic to human tumor cell lines that express the Ag and that the modular design allows a functional exchange of the targeting moiety. Direct comparison with clinically approved Abs demonstrates a superior ability of the bispecific Abs to induce complement-dependent cytotoxicity. The efficacy of the bispecific Abs to activate complement strongly depends on the epitope of the C1q binding Ab, demonstrating that the spatial orientation of the C1 complex upon Ag engagement is a critical factor for efficient complement activation. Collectively, our data provide insight into the mechanism of complement activation and provide a new platform for the development of immunotherapies.

Restriction of C1-inhibitor activity in hereditary angioedema by dominant-negative effects of disease-associated SERPING1 gene variants.

BACKGROUND: Patients with hereditary angioedema experience recurrent, sometimes life-threatening, attacks of edema. It is a rare genetic disorder characterized by genetic and clinical heterogenicity. Most cases are caused by genetic variants in the SERPING1 gene leading to plasma deficiency of the encoded protein C1 inhibitor (C1INH). More than 500 different hereditary angioedema-causing variants have been identified in the SERPING1 gene, but the disease mechanisms by which they result in pathologically low C1INH plasma levels remain largely unknown. OBJECTIVES: The aim was to describe trans-inhibitory effects of full-length or near full-length C1INH encoded by 28 disease-associated SERPING1 variants. METHODS: HeLa cells were transfected with expression constructs encoding the studied SERPING1 variants. Extensive and comparative studies of C1INH expression, secretion, functionality, and intracellular localization were carried out. RESULTS: Our findings characterized functional properties of a subset of SERPING1 variants allowing the examined variants to be subdivided into 5 different clusters, each containing variants sharing specific molecular characteristics. For all variants except 2, we found that coexpression of mutant and normal C1INH negatively affected the overall capacity to target proteases. Strikingly, for a subset of variants, intracellular formation of C1INH foci was detectable only in heterozygous configurations enabling simultaneous expression of normal and mutant C1INH. CONCLUSIONS: We provide a functional classification of SERPING1 gene variants suggesting that different SERPING1 variants drive the pathogenicity through different and in some cases overlapping molecular disease mechanisms. For a subset of gene variants, our data define some types of hereditary angioedema with C1INH deficiency as serpinopathies driven by dominant-negative disease mechanisms.

Inter-α-inhibitor heavy chain H4 and sepsis-related coagulation disturbances: Another link between innate immunity and coagulation.

Background: The protease inhibitor inter-α-inhibitor heavy chain H4 (ITIH4) has been described as an acute-phase reactant and could potentially aid in sepsis monitoring and prognostication. Objectives: To investigate ITIH4 plasma levels in sepsis patients compared with healthy controls and to examine the association between ITIH4 and acute-phase response markers, blood coagulation, and organ dysfunction in sepsis. Methods: We performed a post hoc study to a prospective cohort study. Patients with septic shock (n = 39) were enrolled upon intensive care unit admission. ITIH4 was analyzed using an in-house immunoassay. Standard coagulation parameters, thrombin generation, fibrin formation and lysis, C-reactive protein, organ dysfunction markers, Sequential Organ Failure Assessment score, and disseminated intravascular coagulation (DIC) score were registered. ITIH4 levels were also investigated in a murine Escherichia coli sepsis model. Results: ITIH4 did not display acute-phase behavior as mean ITIH4 levels were not increased in patients with septic shock or in E. coli-infected mice. However, ITIH4 exhibited large interindividual variation in patients with septic shock compared with healthy controls. Low ITIH4 was associated with sepsis-related coagulopathy, including a high DIC score (mean ITIH4: DIC, 203 µg/mL vs non-DIC, 267 µg/mL, P = .01), low antithrombin (r = 0.70, P < .0001) and decreased thrombin generation (mean ITIH4: first peak thrombin tertile, 210 µg/mL vs third peak thrombin tertile, 303 µg/mL, P = .01). ITIH4 showed moderate correlation with arterial blood lactate (ρ = -0.50, P < .001) but only weak correlations with C-reactive protein, alanine transaminase, bilirubin, and Sequential Organ Failure Assessment score (all, ρ < 0.26, P > .05). Conclusion: ITIH4 is associated with sepsis-related coagulopathy but is not an acute-phase reactant during septic shock.

Antigen footprint governs activation of the B cell receptor.

Antigen binding by B cell receptors (BCR) on cognate B cells elicits a response that eventually leads to production of antibodies. However, it is unclear what the distribution of BCRs is on the naïve B cell and how antigen binding triggers the first step in BCR signaling. Using DNA-PAINT super-resolution microscopy, we find that most BCRs are present as monomers, dimers, or loosely associated clusters on resting B cells, with a nearest-neighbor inter-Fab distance of 20-30 nm. We leverage a Holliday junction nanoscaffold to engineer monodisperse model antigens with precision-controlled affinity and valency, and find that the antigen exerts agonistic effects on the BCR as a function of increasing affinity and avidity. Monovalent macromolecular antigens can activate the BCR at high concentrations, whereas micromolecular antigens cannot, demonstrating that antigen binding does not directly drive activation. Based on this, we propose a BCR activation model determined by the antigen footprint.

Highly Increased Levels of Inter-α-inhibitor Heavy Chain 4 (ITIH4) in Autoimmune Cholestatic Liver Diseases.

Background and Aims: There is an unmet need for new biomarkers to improve diagnostics and prognostics in primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC). Inter-α-inhibitor heavy chain 4 (ITIH4) is an abundant, liver-produced protein, and its synthesis may be altered in liver diseases. We investigated whether ITIH4 plasma concentrations were affected in PBC and PSC patients. Methods: We developed an immunoassay specific for ITIH4 and determined ITIH4 plasma concentrations in 66 PBC, 126 PSC, 92 autoimmune hepatitis (AIH), 67 chronic hepatitis C (CHC), 33 alcoholic hepatitis (AH) patients and 138 healthy controls (HCs). Hepatic ITIH4 expression was investigated by immunohistochemistry in PBC. Results: The mean plasma concentration of ITIH4 was almost doubled in PBC [409 µg/mL (95% CI: 388-431)] and 35% higher in PSC [308 µg/mL, (95% CI: 296-319)] compared with HCs [226 µg/mL (95% CI: 221-231); p<0.001]. In PBC patients, ITIH4 correlated with IgM (rho=0.49, p<0.001). Responders to ursodeoxycholic acid treatment (UDCA) had lower levels of ITIH4 than incomplete responders [395 µg/mL (95% CI: 364-425)] vs. 460 µg/mL (95% CI: 421-498); p=0.02]. Four weeks of UDCA treatment had no effect (p=0.19). Increased ITIH4 immunohistochemical staining was seen in a liver biopsy from a PBC patient. ITIH4 levels in AIH [224 µg/mL (95% CI: 208-241)] and HCs were similar (p=0.8). ITIH4 levels were lower in AH [199 µg/mL (95% CI: 175-223)] and CHC [202 µg/mL (192-212)] patients than in HCs (p<0.05). Conclusions: The plasma concentration of ITIH4 was highly elevated in patients with PBC and PSC, suggesting that ITIH4 should be further investigated as a biomarker in cholestatic liver disease.

Mannan-binding lectin serine protease-2 (MASP-2) in human kidney and its relevance for proteolytic activation of the epithelial sodium channel.

Proteolytic activation of the renal epithelial sodium channel (ENaC) is increased by aldosterone. The aldosterone-sensitive protease remains unidentified. In humans, elevated circulating aldosterone is associated with increased urinary extracellular vesicle (uEVs) excretion of mannan-binding lectin associated serine protease-2 (MASP-2). We hypothesized that MASP-2 is a physiologically relevant ENaC-activating protease. It was confirmed that MASP2 mRNA is abundantly present in liver but not in human and mouse kidneys. Aldosterone-stimulation of murine cortical colleting duct (mCCD) cells did not induce MASP-2 mRNA. In human kidney collecting duct, MASP-2 protein was detected in AQP2-negative/ATP6VB1-positive intercalated cells suggestive of MASP2 protein uptake. Plasma concentration of full-length MASP-2 and the short splice variant MAp19 were not changed in a cross-over intervention study in healthy humans with low (70 mmol/day) versus high (250 mmol/day) Na+ intake despite changes in aldosterone. The ratio of MAp19/MASP-2 in plasma was significantly increased with a high Na+ diet and the ratio correlated with changes in aldosterone and fractional Na+ excretion. MASP-2 was not detected in crude urine or in uEVs. MASP2 activated an amiloride-sensitive current when co-expressed with ENaC in Xenopus oocytes, but not when added to the bath solution. In monolayers of collecting duct M1 cells, MASP2 expression did not increase amiloride-sensitive current and in HEK293 cells, MASP-2 did not affect γENaC cleavage. MASP-2 is neither expressed nor co-localized and co-regulated with ENaC in the human kidney or in urine after low Na+ intake. MASP-2 does not mediate physiological ENaC cleavage in low salt/high aldosterone settings.

Novel homozygous CD46 variant with C-isoform expression affects C3b inactivation in atypical hemolytic uremic syndrome.

Atypical hemolytic uremic syndrome (aHUS) is a thrombotic microangiopathy that may lead to organ failure. Dysregulation of the complement system can cause aHUS, and various disease-related variants in the complement regulatory protein CD46 are described. We here report a pediatric patient with aHUS carrying a hitherto unreported homozygous variant in CD46 (NM_172359.3:c.602C>T p.(Ser201Leu)). In our functional analyses, this variant caused complement dysregulation through three separate mechanisms. First, CD46 surface expression on the patient's blood cells was significantly reduced. Second, stably expressing CD46(Ser201Leu) cells bound markedly less to patterns of C3b than CD46 WT cells. Third, the patient predominantly expressed the rare isoforms of CD46 (C dominated) instead of the more common isoforms (BC dominated). Using BC1 and C1 expressing cell lines, we found that the C1 isoform bound markedly less C3b than the BC1 isoform. These results highlight the coexistence of multiple mechanisms that may act synergistically to disrupt CD46 function during aHUS development.

Structure-Guided Engineering of a Complement Component C3-Binding Nanobody Improves Specificity and Adds Cofactor Activity.

The complement system is a part of the innate immune system, where it labels intruding pathogens as well as dying host cells for clearance. If complement regulation is compromised, the system may contribute to pathogenesis. The proteolytic fragment C3b of complement component C3, is the pivot point of the complement system and provides a scaffold for the assembly of the alternative pathway C3 convertase that greatly amplifies the initial complement activation. This makes C3b an attractive therapeutic target. We previously described a nanobody, hC3Nb1 binding to C3 and its degradation products. Here we show, that extending the N-terminus of hC3Nb1 by a Glu-Trp-Glu motif renders the resulting EWE-hC3Nb1 (EWE) nanobody specific for C3 degradation products. By fusing EWE to N-terminal CCP domains from complement Factor H (FH), we generated the fusion proteins EWEnH and EWEµH. In contrast to EWE, these fusion proteins supported Factor I (FI)-mediated cleavage of human and rat C3b. The EWE, EWEµH, and EWEnH proteins bound C3b and iC3b with low nanomolar dissociation constants and exerted strong inhibition of alternative pathway-mediated deposition of complement. Interestingly, EWEnH remained soluble above 20 mg/mL. Combined with the observed reactivity with both human and rat C3b as well as the ability to support FI-mediated cleavage of C3b, this features EWEnH as a promising candidate for in vivo studies in rodent models of complement driven pathogenesis.

A low level of naturally occurring antibodies associates with functional antibody deficiency.

Functional antibody deficiency is clinically assessed from antibody responses to vaccination. However, diagnostic vaccination is complex and may fail in practice. We hypothesized that the levels of naturally occurring antibodies against galactose-α-1,3-galactose (αGal) may represent alternative markers of functional antibody capacity. We included data from 229 patients with suspected primary immunodeficiency in a retrospective study. Antibody levels against αGal and twelve pneumococcal serotypes were determined with solid-phase immunoassays. Pneumococcal vaccinations and treatment with normal human immunoglobulin were assessed from medical records. Anti-αGal antibody levels correlated positively with anti-pneumococcal antibody levels measured before and after pneumococcal vaccination. Contrary to the anti-pneumococcal antibody levels, the anti-αGal antibody level showed potential for predicting subsequent immunoglobulin treatment - a marker of disease severity. Naturally occurring antibodies may reflect the functional capacity tested by diagnostic vaccination but add more useful clinical data. The clinical utility of this easy test should be evaluated in prospective studies.