Inhibition of the C1s Protease and the Classical Complement Pathway by 6-(4-Phenylpiperazin-1-yl)Pyridine-3-Carboximidamide and Chemical Analogs.

The classical pathway (CP) is a potent mechanism for initiating complement activity and is a driver of pathology in many complement-mediated diseases. The CP is initiated via activation of complement component C1, which consists of the pattern recognition molecule C1q bound to a tetrameric assembly of proteases C1r and C1s. Enzymatically active C1s provides the catalytic basis for cleavage of the downstream CP components, C4 and C2, and is therefore an attractive target for therapeutic intervention in CP-driven diseases. Although an anti-C1s mAb has been Food and Drug Administration approved, identifying small-molecule C1s inhibitors remains a priority. In this study, we describe 6-(4-phenylpiperazin-1-yl)pyridine-3-carboximidamide (A1) as a selective, competitive inhibitor of C1s. A1 was identified through a virtual screen for small molecules that interact with the C1s substrate recognition site. Subsequent functional studies revealed that A1 dose-dependently inhibits CP activation by heparin-induced immune complexes, CP-driven lysis of Ab-sensitized sheep erythrocytes, CP activation in a pathway-specific ELISA, and cleavage of C2 by C1s. Biochemical experiments demonstrated that A1 binds directly to C1s with a Kd of ∼9.8 µM and competitively inhibits its activity with an inhibition constant (Ki) of ∼5.8 µM. A 1.8-Å-resolution crystal structure revealed the physical basis for C1s inhibition by A1 and provided information on the structure-activity relationship of the A1 scaffold, which was supported by evaluating a panel of A1 analogs. Taken together, our work identifies A1 as a new class of small-molecule C1s inhibitor and lays the foundation for development of increasingly potent and selective A1 analogs for both research and therapeutic purposes.

The axis of complement C1 and nucleolus in antinuclear autoimmunity.

Antinuclear autoantibodies (ANA) are heterogeneous self-reactive antibodies that target the chromatin network, the speckled, the nucleoli, and other nuclear regions. The immunological aberration for ANA production remains partially understood, but ANA are known to be pathogenic, especially, in systemic lupus erythematosus (SLE). Most SLE patients exhibit a highly polygenic disease involving multiple organs, but in rare complement C1q, C1r, or C1s deficiencies, the disease can become largely monogenic. Increasing evidence point to intrinsic autoimmunogenicity of the nuclei. Necrotic cells release fragmented chromatins as nucleosomes and the alarmin HMGB1 is associated with the nucleosomes to activate TLRs and confer anti-chromatin autoimmunogenecity. In speckled regions, the major ANA targets Sm/RNP and SSA/Ro contain snRNAs that confer autoimmunogenecity to Sm/RNP and SSA/Ro antigens. Recently, three GAR/RGG-containing alarmins have been identified in the nucleolus that helps explain its high autoimmunogenicity. Interestingly, C1q binds to the nucleoli exposed by necrotic cells to cause protease C1r and C1s activation. C1s cleaves HMGB1 to inactive its alarmin activity. C1 proteases also degrade many nucleolar autoantigens including nucleolin, a major GAR/RGG-containing autoantigen and alarmin. It appears that the different nuclear regions are intrinsically autoimmunogenic by containing autoantigens and alarmins. However, the extracellular complement C1 complex function to dampen nuclear autoimmunogenecity by degrading these nuclear proteins.

The association between complement C1q tumour necrosis factor-related protein-1 (CTRP-1) level and metabolic syndrome.

INTRODUCTION: Complement C1q tumour necrosis factor-related protein (CTRP-1) is a member of the C1q protein superfamily that plays a role in metabolism. This retrospective study aimed to investigate associations between CTRP-1 and metabolic syndrome (MetS). MATERIAL AND METHODS: This study screened subjects who had undergone regular health examinations at the Physical Examination Centre in the First People's Hospital of Yinchuan (the Second Affiliated Hospital of Ningxia Medical University) between November 2017 and September 2020. The total recruited population included 430 subjects who had undergone regular health examinations, excluding 112 subjects with high glycated haemoglobin (HbA1c ≥ 7). Finally, the data of 318 participants were further analysed. Non-diabetic subjects were divided into 2 groups: one with MetS and one without MetS (controls). Serum CTRP-1 concentrations were evaluated using an enzyme-linked immunosorbent assay. RESULTS: A total of 318 subjects were included, among whom 176 were diagnosed with MetS (MetS group) and 142 were not (non-MetS controls). The MetS group had significantly lower CTRP-1 levels than non-MetS controls (128.51 [111.56-143.05] vs. 138.82 [122.83-154.33] ng/mL, p < 0.001). Correlation analysis showed that serum CTRP-1 levels correlated negatively with body mass index (r = -0.161, p = 0.004), waist circumference (r = -0.191, p = 0.001), systolic blood pressure (r = -0.198, p < 0.001), diastolic blood pressure (r = -0.145, p = 0.010), fasting blood glucose (FBG) (r = -0.562, p < 0.001), fasting insulin (FIns) (r = -0.424, p < 0.001), and homeostasis model assessment of insulin resistance (HOMA-IR) (r = -0.541, p < 0.001). Multiple linear regression models showed that CTRP-1 levels were associated with MetS (p < 0.01). The lipid profile area under the curve (AUC) was comparable to those for FBG and FIns, and it was significantly higher than the AUCs for demographic variables. CONCLUSIONS: The results of this study suggest that the serum CTRP-1 level is negatively associated with MetS. CTRP-1 is a potential metabolism-related protein and is likely to be associated with lipid profiles in MetS.

C9-ALS-Associated Proline-Arginine Dipeptide Repeat Protein Induces Activation of NLRP3 Inflammasome of HMC3 Microglia Cells by Binding of Complement Component 1 Q Subcomponent-Binding Protein (C1QBP), and Syringin Prevents This Effect.

Amyotrophic lateral sclerosis (ALS) is a fatal disease in which motor neurons gradually degenerate. The mutation of the C9orf72 gene is the main genetic cause of ALS (C9-ALS). One of its specific pathological features is the production of proline-arginine (PR) dipeptide repeat protein (DPR). In this study, we developed a PR-DPR (PR50)-expressing human HMC3 microglial cell model. We found that PR50 mainly aggregates into spots in the nucleus and induces significant NLRP3 inflammasome activity. Moreover, mouse NSC-34 motor neuron cells treated with a conditional medium of PR50-expressing HMC3 cells (PR-CM) caused cell damage and apoptosis activity. However, R50-expressing HMC cells treated with MCC950 (an NLRP3 inhibitor) reversed this result. Furthermore, we identified complement component 1 q subcomponent-binding protein (C1QBP) as one of the interaction partners of PR50. The downregulation of C1QBP in HMC3 cells induces NLRP3 inflammasome activity similar to PR50 expression. Finally, we found that syringin can block the interaction between PR50 and C1QBP, and effectively reduce the PR50-induced NLRP3 inflammasome activity in HMC3 cells. This improves the apoptosis of NSC-34 cells caused by PR-CM. This study is the first to link PR50, C1QBP, and NLRP3 inflammasome activity in microglia and develop potential therapeutic strategies for syringin intervention in C9-ALS.

Toward Understanding How Staphylococcal Protein A Inhibits IgG-Mediated Phagocytosis.

IgG molecules are crucial for the human immune response against bacterial infections. IgGs can trigger phagocytosis by innate immune cells, like neutrophils. To do so, IgGs should bind to the bacterial surface via their variable Fab regions and interact with Fcγ receptors and complement C1 via the constant Fc domain. C1 binding to IgG-labeled bacteria activates the complement cascade, which results in bacterial decoration with C3-derived molecules that are recognized by complement receptors on neutrophils. Next to FcγRs and complement receptors on the membrane, neutrophils also express the intracellular neonatal Fc receptor (FcRn). We previously reported that staphylococcal protein A (SpA), a key immune-evasion protein of Staphylococcus aureus, potently blocks IgG-mediated complement activation and killing of S. aureus by interfering with IgG hexamer formation. SpA is also known to block IgG-mediated phagocytosis in absence of complement, but the mechanism behind it remains unclear. In this study, we demonstrate that SpA blocks IgG-mediated phagocytosis and killing of S. aureus and that it inhibits the interaction of IgGs with FcγRs (FcγRIIa and FcγRIIIb, but not FcγRI) and FcRn. Furthermore, our data show that multiple SpA domains are needed to effectively block IgG1-mediated phagocytosis. This provides a rationale for the fact that SpA from S. aureus contains four to five repeats. Taken together, our study elucidates the molecular mechanism by which SpA blocks IgG-mediated phagocytosis and supports the idea that in addition to FcγRs, the intracellular FcRn is also prevented from binding IgG by SpA.

CTRP11 contributes modestly to systemic metabolism and energy balance.

C1q/TNF-related proteins (CTRP1-15) constitute a conserved group of secreted proteins of the C1q family with diverse functions. In vitro studies have shown that CTRP11/C1QL4 can inhibit adipogenesis, antagonize myoblast fusion, and promote testosterone synthesis and secretion. Whether CTRP11 is required for these processes in vivo remains unknown. Here, we show that knockout (KO) mice lacking CTRP11 have normal skeletal muscle mass and function, and testosterone level, suggesting that CTRP11 is dispensable for skeletal muscle development and testosterone production. We focused our analysis on whether this nutrient-responsive secreted protein plays a role in controlling sugar and fat metabolism. At baseline when mice are fed a standard chow, CTRP11 deficiency affects metabolic parameters in a sexually dimorphic manner. Only Ctrp11-KO female mice have significantly higher fasting serum ketones and reduced physical activity. In the refeeding phase following food withdrawal, Ctrp11-KO female mice have reduced food intake and increased metabolic rate and energy expenditure, highlighting CTRP11's role in fasting-refeeding response. When challenged with a high-fat diet to induce obesity and metabolic dysfunction, CTRP11 deficiency modestly exacerbates obesity-induced glucose intolerance, with more pronounced effects seen in Ctrp11-KO male mice. Switching to a low-fat diet after obesity induction results in greater fat loss in wild type relative to KO male mice, suggesting impaired response to obesity reversal and reduced metabolic flexibility in the absence of CTRP11. Collectively, our data provide genetic evidence for novel sex-dependent metabolic regulation by CTRP11, but note the overall modest contribution of CTRP11 to systemic energy homeostasis.

Quantitative Visualization of the Interaction between Complement Component C1 and Immunoglobulin G: The Effect of CH1 Domain Deletion.

Immunoglobulin G (IgG) adopts a modular multidomain structure that mediates antigen recognition and effector functions, such as complement-dependent cytotoxicity. IgG molecules are self-assembled into a hexameric ring on antigen-containing membranes, recruiting the complement component C1q. In order to provide deeper insights into the initial step of the complement pathway, we report a high-speed atomic force microscopy study for the quantitative visualization of the interaction between mouse IgG and the C1 complex composed of C1q, C1r, and C1s. The results showed that the C1q in the C1 complex is restricted regarding internal motion, and that it has a stronger binding affinity for on-membrane IgG2b assemblages than C1q alone, presumably because of the lower conformational entropy loss upon binding. Furthermore, we visualized a 1:1 stoichiometric interaction between C1/C1q and an IgG2a variant that lacks the entire CH1 domain in the absence of an antigen. In addition to the canonical C1q-binding site on Fc, their interactions are mediated through a secondary site on the CL domain that is cryptic in the presence of the CH1 domain. Our findings offer clues for novel-modality therapeutic antibodies.

Expression of immune-related genes as prognostic biomarkers for the assessment of osteosarcoma clinical outcomes.

Cancer immunotherapy is a promising therapeutic approach, but the prognostic value of immune-related genes in osteosarcoma (OS) is unknown. Here, Target-OS RNA-seq data were analyzed to detect differentially expressed genes (DEGs) between OS subgroups, followed by functional enrichment analysis. Cox proportional risk regression was performed for each immune-related gene, and a risk score model to predict the prognosis of patients with OS was constructed. The risk scores were calculated using the risk signature to divide the training set into high-risk and low-risk groups, and validation was performed with GSE21257. We identified two immune-associated clusters, C1 and C2. C1 was closely related to immunity, and the immune score was significantly higher in C1 than in C2. Furthermore, we validated 6 immune cell hub genes related to the prognosis of OS: CD8A, KIR2DL1, CD79A, APBB1IP, GAL, and PLD3. Survival analysis revealed that the prognosis of the high-risk group was significantly worse than that of the low-risk group. We also explored whether the 6-gene prognostic risk model was effective for survival prediction. In conclusion, the constructed a risk score model based on immune-related genes and the survival of patients with OS could be a potential tool for targeted therapy.

Where we are with acquired angioedema due to C1 inhibitor deficiency: A systematic literature review.

Acquired angioedema due to C1 inhibitor deficiency (C1-INH-AAE) is a rare disease characterized by adult-onset recurrent non-urticarial angioedema with low levels of C1-INH. It is associated with lymphoproliferative disorders, and treatments are off-label with variable success. We conducted a systematic literature review to include patients with C1-INH-AAE identified in PubMed and Embase databases between January 2006 and February 2021. Clinical features of these patients were summarized, and factors associated with disease remission were explored. A total of 121 patients were included in the current study with a median age at diagnosis of 64 years and 45.5% being male. An associated disease was recorded in 94 patients (77.7%), and lymphoproliferative disorder was the most reported (59/94, 62.8%). Anti-C1-INH autoantibodies were present in 45 of 71 patients (63.4%). Factors impacting disease remissions included age (odds ratio [OR] 0.951, 95% confidence interval [CI] 0.909-0.994, p = 0.027), male (OR 0.327, 95% CI 0.124-0.866, p = 0.025), presence of monoclonal gammopathy (OR 0.133, 95% CI 0.041-0.429, p = 0.001), requirement of specific on-demand treatment (OR 0.216, 95% CI 0.066-0.709, p = 0.012) and rituximab use (OR 2.865, 95% CI 1.038-7.911, p = 0.042). A total of nine patients (7.4%) died at last follow up and most were unrelated to C1-INH-AAE. Our results imply that C1-INH-AAE is primarily associated with underlying B or plasma cell abnormalities, and clone-directed therapies could be promising options for its long-term management.

Complement C1s cleaves PfEMP1 at interdomain conserved sites inhibiting Plasmodium falciparum cytoadherence.

Cytoadhesion of Plasmodium falciparum-infected erythrocytes (IEs) to the endothelial lining of blood vessels protects parasites from splenic destruction, but also leads to detrimental inflammation and vessel occlusion. Surface display of the P. falciparum erythrocyte membrane protein 1 (PfEMP1) adhesion ligands exposes them to host antibodies and serum proteins. PfEMP1 are important targets of acquired immunity to malaria, and through evolution, the protein family has expanded and diversified to bind a select set of host receptors through antigenically diversified receptor-binding domains. Here, we show that complement component 1s (C1s) in serum cleaves PfEMP1 at semiconserved arginine motifs located at interdomain regions between the receptor-binding domains, rendering the IE incapable of binding the two main PfEMP1 receptors, CD36 and endothelial protein C receptor (EPCR). Bioinformatic analyses of PfEMP1 protein sequences from 15 P. falciparum genomes found the C1s motif was present in most PfEMP1 variants. Prediction of C1s cleavage and loss of binding to endothelial receptors was further corroborated by testing of several different parasite lines. These observations suggest that the parasites have maintained susceptibility for cleavage by the serine protease, C1s, and provides evidence for a complex relationship between the complement system and the P. falciparum cytoadhesion virulence determinant.

Nanobodies Provide Insight into the Molecular Mechanisms of the Complement Cascade and Offer New Therapeutic Strategies.

The complement system is part of the innate immune response, where it provides immediate protection from infectious agents and plays a fundamental role in homeostasis. Complement dysregulation occurs in several diseases, where the tightly regulated proteolytic cascade turns offensive. Prominent examples are atypical hemolytic uremic syndrome, paroxysmal nocturnal hemoglobinuria and Alzheimer's disease. Therapeutic intervention targeting complement activation may allow treatment of such debilitating diseases. In this review, we describe a panel of complement targeting nanobodies that allow modulation at different steps of the proteolytic cascade, from the activation of the C1 complex in the classical pathway to formation of the C5 convertase in the terminal pathway. Thorough structural and functional characterization has provided a deep mechanistic understanding of the mode of inhibition for each of the nanobodies. These complement specific nanobodies are novel powerful probes for basic research and offer new opportunities for in vivo complement modulation.

Initiators of Classical and Lectin Complement Pathways Are Differently Engaged after Traumatic Brain Injury-Time-Dependent Changes in the Cortex, Striatum, Thalamus and Hippocampus in a Mouse Model.

The complement system is involved in promoting secondary injury after traumatic brain injury (TBI), but the roles of the classical and lectin pathways leading to complement activation need to be clarified. To this end, we aimed to determine the ability of the brain to activate the synthesis of classical and lectin pathway initiators in response to TBI and to examine their expression in primary microglial cell cultures. We have modeled TBI in mice by controlled cortical impact (CCI), a clinically relevant experimental model. Using Real-time quantitative polymerase chain reaction (RT-qPCR) we analyzed the expression of initiators of classical the complement component 1q, 1r and 1s (C1q, C1r, and C1s) and lectin (mannose binding lectin A, mannose binding lectin C, collectin 11, ficolin A, and ficolin B) complement pathways and other cellular markers in four brain areas (cortex, striatum, thalamus and hippocampus) of mice exposed to CCI from 24 h and up to 5 weeks. In all murine ipsilateral brain structures assessed, we detected long-lasting, time- and area-dependent significant increases in the mRNA levels of all classical (C1q, C1s, C1r) and some lectin (collectin 11, ficolin A, ficolin B) initiator molecules after TBI. In parallel, we observed significantly enhanced expression of cellular markers for neutrophils (Cd177), T cells (Cd8), astrocytes (glial fibrillary acidic protein-GFAP), microglia/macrophages (allograft inflammatory factor 1-IBA-1), and microglia (transmembrane protein 119-TMEM119); moreover, we detected astrocytes (GFAP) and microglia/macrophages (IBA-1) protein level strong upregulation in all analyzed brain areas. Further, the results obtained in primary microglial cell cultures suggested that these cells may be largely responsible for the biosynthesis of classical pathway initiators. However, microglia are unlikely to be responsible for the production of the lectin pathway initiators. Immunofluorescence analysis confirmed that at the site of brain injury, the C1q is localized in microglia/macrophages and neurons but not in astroglial cells. In sum, the brain strongly reacts to TBI by activating the local synthesis of classical and lectin complement pathway activators. Thus, the brain responds to TBI with a strong, widespread and persistent upregulation of complement components, the targeting of which may provide protection in TBI.

Treatment of COVID-19 With Conestat Alfa, a Regulator of the Complement, Contact Activation and Kallikrein-Kinin System.

A dysregulated immune response with hyperinflammation is observed in patients with severe coronavirus disease 2019 (COVID-19). The aim of the present study was to assess the safety and potential benefits of human recombinant C1 esterase inhibitor (conestat alfa), a complement, contact activation and kallikrein-kinin system regulator, in severe COVID-19. Patients with evidence of progressive disease after 24 h including an oxygen saturation <93% at rest in ambient air were included at the University Hospital Basel, Switzerland in April 2020. Conestat alfa was administered by intravenous injections of 8400 IU followed by 3 additional doses of 4200 IU in 12-h intervals. Five patients (age range, 53-85 years; one woman) with severe COVID-19 pneumonia (11-39% lung involvement on computed tomography scan of the chest) were treated a median of 1 day (range 1-7 days) after admission. Treatment was well-tolerated. Immediate defervescence occurred, and inflammatory markers and oxygen supplementation decreased or stabilized in 4 patients (e.g., median C-reactive protein 203 (range 31-235) mg/L before vs. 32 (12-72) mg/L on day 5). Only one patient required mechanical ventilation. All patients recovered. C1INH concentrations were elevated before conestat alfa treatment. Levels of complement activation products declined after treatment. Viral loads in nasopharyngeal swabs declined in 4 patients. In this uncontrolled case series, targeting multiple inflammatory cascades by conestat alfa was safe and associated with clinical improvements in the majority of severe COVID-19 patients. Controlled clinical trials are needed to assess its safety and efficacy in preventing disease progression.

The Interactions of Parasite Calreticulin With Initial Complement Components: Consequences in Immunity and Virulence.

Because of its capacity to increase a physiologic inflammatory response, to stimulate phagocytosis, to promote cell lysis and to enhance pathogen immunogenicity, the complement system is a crucial component of both the innate and adaptive immune responses. However, many infectious agents resist the activation of this system by expressing or secreting proteins with a role as complement regulatory, mainly inhibitory, proteins. Trypanosoma cruzi, the causal agent of Chagas disease, a reemerging microbial ailment, possesses several virulence factors with capacity to inhibit complement at different stages of activation. T. cruzi calreticulin (TcCalr) is a highly-conserved, endoplasmic reticulum-resident chaperone that the parasite translocates to the extracellular environment, where it exerts a variety of functions. Among these functions, TcCalr binds C1, MBL and ficolins, thus inhibiting the classical and lectin pathways of complement at their earliest stages of activation. Moreover, the TcCalr/C1 interaction also mediates infectivity by mimicking a strategy used by apoptotic cells for their removal. More recently, it has been determined that these Calr strategies are also used by a variety of other parasites. In addition, as reviewed elsewhere, TcCalr inhibits angiogenesis, promotes wound healing and reduces tumor growth. Complement C1 is also involved in some of these properties. Knowledge on the role of virulence factors, such as TcCalr, and their interactions with complement components in host-parasite interactions, may lead toward the description of new anti-parasite therapies and prophylaxis.

A first of its kind quantitative functional C1-esterase inhibitor lateral flow assay for hereditary angioedema point-of-care diagnostic testing.

Hereditary Angioedema (HAE) is a rare, autosomal dominant disease caused by mutations in SERPING1 gene leading to dysfunction/deficiency of C1-esterase inhibitor (C1-INH) protein and subsequent dysregulation of the contact system and bradykinin overproduction. As functional C1-INH (fC1-INH) levels are reduced in HAE types I and II (HAE-I/II), a specific, sensitive and accessible rapid diagnostic method to quantitate fC1-INH is crucial in diagnosing HAE-I/II. Previously, we developed/validated methods to detect fC1-INH levels in human plasma based on functional binding to C1s or FXIIa for C1-INH-based therapies. Quantitative fC1-INH immunoassay methods were converted to the Lateral flow assay (LFA) platform after identifying the best reagent/s pair. The assay was developed and optimized as a first of its kind LFA method for quantifying fC1-INH in human plasma to aid HAE point-of-care diagnosis. Receiver operating characteristic analysis was performed using normal control and HAE subject plasma samples to calculate area-under-curve and a cut-off point to distinguish normal versus HAE subject samples. LFA data was correlated with the conventional diagnostic assay for fC1-INH in HAE plasma samples and profiles matched for individual subjects. Here, we demonstrate a proof-of-concept for the quantitative fC1-INH LFA using normal and HAE plasma samples. We propose that the method could be used as a point-of-care test to diagnose HAE in a variety of settings, such as, a hospital or physician's office, at home or in an ambulance.

Extracellular Vesicle-Induced Classical Complement Activation Leads to Retinal Endothelial Cell Damage via MAC Deposition.

Several studies have suggested that there is a link between membrane attack complex (MAC) deposition in the retina and the progression of diabetic retinopathy (DR). Our recent investigation demonstrated that circulating IgG-laden extracellular vesicles contribute to an increase in retinal vascular permeability in DR through activation of the complement system. However, the mechanism through which extracellular vesicle-induced complement activation contributes to retinal vascular cytolytic damage in DR is not well understood. In this study, we demonstrate that IgG-laden extracellular vesicles in rat plasma activate the classical complement pathway, and in vitro Streptozotocin (STZ)-induced rat diabetic plasma results in MAC deposition and cytolytic damage in human retinal endothelial cells (HRECs). Moreover, removal of the plasma extracellular vesicles reduced the MAC deposition and abrogated cytolytic damage seen in HRECs. Together, the results of this study demonstrate that complement activation by IgG-laden extracellular vesicles in plasma could lead to MAC deposition and contribute to endothelium damage and progression of DR.

[Nephropathy associated with hypocomplementemic urticarial vasculitis: A case report and literature review]. / Néphropathie associée à une vascularite urticarienne hypocomplémentémique : présentation d'un cas clinique et revue de la littérature.

Hypocomplementemic urticarial vasculitis is a rare systemic vasculitis, affecting small vessels, characterised by chronicle urticaria, hypocomplementemia, and systemic manifestations. Renal involvement, whose prevalence varies between 9% and 60%, is mainly glomerular. We here report the case of a 59 years old woman presenting kidney failure, associated with chronicle urticaria and arthralgias. Laboratory investigation showed haematuria, proteinuria, hypocomplementemia and anti-SSa antibody positivity. A percutaneous kidney biopsy revealed focal and segmental glomerulonephritis associated with an acute interstitial nephritis. Hypocomplementemic urticarial vasculitis diagnosis was established after identifying anti-C1q antibodies. The lack of a dry syndrome, the negativity of a Schirmer test and the lack of sialadenitis on a salivary gland biopsy excluded an associated Gougerot-Sjögren Syndrome. The patient was treated with hydroxychloroquine and low-dose steroids, enabling a clinical and biological recovery. Of the 82 cases in the literature describing hypocomplementemic urticarial vasculitis associated nephropathies, 72 (88%) were a glomerular impairment, most frequently secondary to membranoproliferative glomerulonephritis. Only 6 (7%) tubulo-interstitial nephritis have been reported, 4 of them being associated with a glomerulonephritis. Patients were more likely to be women, aged in their third decade. The most frequent renal manifestations were haematuria (60%), and proteinuria (52%). Kidney failure was rarely observed (22%), with a fairly good renal prognosis. Hypocomplementemic urticarial vasculitis was associated with a systemic disease in 11 (13%) patients. In the absence of recommendations, the treatment strategy remains to be defined.

C1R Mutations Trigger Constitutive Complement 1 Activation in Periodontal Ehlers-Danlos Syndrome.

Heterozygous missense or in-frame insertion/deletion mutations in complement 1 subunits C1r and C1s cause periodontal Ehlers-Danlos Syndrome (pEDS), a specific EDS subtype characterized by early severe periodontal destruction and connective tissue abnormalities like easy bruising, pretibial haemosiderotic plaques, and joint hypermobility. We report extensive functional studies of 16 C1R variants associated with pEDS by in-vitro overexpression studies in HEK293T cells followed by western blot, size exclusion chromatography and surface plasmon resonance analyses. Patient-derived skin fibroblasts were analyzed by western blot and Enzyme-linked Immunosorbent Assay (ELISA). Overexpression of C1R variants in HEK293T cells revealed that none of the pEDS variants was integrated into the C1 complex but cause extracellular presence of catalytic C1r/C1s activities. Variants showed domain-specific abnormalities of intracellular processing and secretion with preservation of serine protease function in the supernatant. In contrast to C1r wild type, and with the exception of a C1R missense variant disabling a C1q binding site, pEDS variants had different impact on the cell: retention of C1r fragments inside the cell, secretion of aggregates, or a new C1r cleavage site. Overexpression of C1R variants in HEK293T as well as western blot analyses of patient fibroblasts showed decreased levels of secreted C1r. Importantly, all available patient fibroblasts exhibited activated C1s and activation of externally added C4 in the supernatant while control cell lines secreted proenzyme C1s and showed no increase in C4 activation. The central elements in the pathogenesis of pEDS seem to be the intracellular activation of C1r and/or C1s, and extracellular presence of activated C1s that independently of microbial triggers can activate the classical complement cascade.