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International audience; ObjectivesRheumatoid arthritis (RA) is a debilitating disease, but patient management and treatment have been revolutionized since the advent of bDMARDs. However, about one third of RA patients do not respond to specific bDMARD treatment without clear identified reasons. Different bDMARDs must be tried until the right drug is found. Here, we sought to identify a predictive protein signature to stratify patient responsiveness to rituximab (RTX) among patients with an insufficient response to a first anti-TNFα treatment.MethodsSerum samples were collected at baseline before RTX initiation. A proteomics study comparing responders and nonresponders was conducted to identify and select potential predictive biomarkers whose concentration was measured by quantitative assays. Logistic regression was performed to determine the best biomarker combination to predict good or nonresponse to RTX (EULAR criteria after 6 months’ treatment).ResultsEleven biomarkers potentially discriminating between responders and nonresponders were selected following discovery proteomics. Quantitative immunoassays and univariate statistical analysis showed that fetuin-A and thyroxine binding globulin (TBG) presented a good capacity to discriminate between patient groups. A logistic regression analysis revealed that the combination of fetuin-A plus TBG could accurately predict a patient’s responsiveness to RTX with an AUC of 0.86, sensitivity of 80%, and a specificity of 79%.ConclusionIn RA patients for whom a first anti-TNFα treatment has failed, the serum abundance of fetuin-A and TBG before initiating RTX treatment is an indicator for their response status at 6 months. ClinicalTrials.gov identifier: NCT01000441.

COVID-19 is caused by the human pathogen severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and has resulted in widespread morbidity and mortality. CD4+ T cells, CD8+ T cells and neutralizing antibodies all contribute to control SARS-CoV-2 infection. However, heterogeneity is a major factor in disease severity and in immune innate and adaptive responses to SARS-CoV-2. We performed a deep analysis by flow cytometry of lymphocyte populations of 125 hospitalized SARS-CoV-2 infected patients on the day of hospital admission. Five clusters of patients were identified using hierarchical classification on the basis of their immunophenotypic profile, with different mortality outcomes. Some characteristics were observed in all the clusters of patients, such as lymphopenia and an elevated level of effector CD8+CCR7- T cells. However, low levels of T cell activation are associated to a better disease outcome; on the other hand, profound CD8+ T-cell lymphopenia, a high level of CD4+ and CD8+ T-cell activation and a high level of CD8+ T-cell senescence are associated with a higher mortality outcome. Furthermore, a cluster of patient was characterized by high B-cell responses with an extremely high level of plasmablasts. Our study points out the prognostic value of lymphocyte parameters such as T-cell activation and senescence and strengthen the interest in treating the patients early in course of the disease with targeted immunomodulatory therapies based on the type of adaptive response of each patient.

International audience; Our immune system responds to infectious (PAMPs) and tissue damage (DAMPs) signals. The complement system and alarmin High-Mobility Group Box 1 (HMGB1) are two powerful soluble actors of human host defense and immune surveillance. These systems involve molecular cascades and amplification loops for their signaling or activation. Initially activated as alarm raising systems, their function can be finally switched towards inflammation resolution, where they sustain immune maturation and orchestrate repair mechanisms, opening the way back to homeostasis. However, when getting out of control, these defense systems can become deleterious and trigger serious cellular and tissue damage. Therefore, they can be considered as double-edged swords. The close interaction between the complement and HMGB1 pathways is described here, as well as their traditional and non-canonical roles, their functioning at different locations and their independent and collective impact in different systems both in health and disease. Starting from these systems and interplay at the molecular level (when elucidated), we then provide disease examples to better illustrate the signs and consequences of their roles and interaction, highlighting their importance and possible vicious circles in alarm raising and inflammation, both individually or in combination. Although this integrated view may open new therapeutic strategies, future challenges have to be faced because of the remaining unknowns regarding the molecular mechanisms underlying the fragile molecular balance which can drift towards disease or return to homeostasis, as briefly discussed at the end.

BackgroundThe SARS-CoV-2 infection triggers excessive immune response resulting in increased levels of pro-inflammatory cytokines, endothelial injury, and intravascular coagulopathy. The complement system (CS) activation participates to this hyperinflammatory response. However, it is still unclear which activation pathways (classical, alternative, or lectin pathway) pilots the effector mechanisms that contribute to critical illness. To better understand the immune correlates of disease severity, we performed an analysis of CS activation pathways and components in samples collected from COVID-19 patients hospitalized in Grenoble Alpes University Hospital between 1 and 30 April 2020 and of their relationship with the clinical outcomes.MethodsWe conducted a retrospective, single-center study cohort in 74 hospitalized patients with RT-PCR-proven COVID-19. The functional activities of classical, alternative, and mannose-binding lectin (MBL) pathways and the antigenic levels of the individual components C1q, C4, C3, C5, Factor B, and MBL were measured in patients’ samples during hospital admission. Hierarchical clustering with the Ward method was performed in order to identify clusters of patients with similar characteristics of complement markers. Age was included in the model. Then, the clusters were compared with the patient clinical features: rate of intensive care unit (ICU) admission, corticoid treatment, oxygen requirement, and mortality.ResultsFour clusters were identified according to complement parameters. Among them, two clusters revealed remarkable profiles: in one cluster (n = 15), patients exhibited activation of alternative and lectin pathways and low antigenic levels of MBL, C4, C3, Factor B, and C5 compared to all the other clusters; this cluster had the higher proportion of patients who died (27%) and required oxygen support (80%) or ICU care (53%). In contrast, the second cluster (n = 19) presented inflammatory profile with high classical pathway activity and antigenic levels of complement components; a low proportion of patients required ICU care (26%) and no patient died in this group.ConclusionThese findings argue in favor of prominent activation of the alternative and MBL complement pathways in severe COVID-19, but the spectrum of complement involvement seems to be heterogeneous requiring larger studies.

COVID-19 is caused by the human pathogen severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and has resulted in widespread morbidity and mortality. CD4+ T cells, CD8+ T cells and neutralizing antibodies all contribute to control SARS-CoV-2 infection. However, heterogeneity is a major factor in disease severity and in immune innate and adaptive responses to SARS-CoV-2. We performed a deep analysis by flow cytometry of lymphocyte populations of 125 hospitalized SARS-CoV-2 infected patients on the day of hospital admission. Five clusters of patients were identified using hierarchical classification on the basis of their immunophenotypic profile, with different mortality outcomes. Some characteristics were observed in all the clusters of patients, such as lymphopenia and an elevated level of effector CD8+CCR7- T cells. However, low levels of T cell activation are associated to a better disease outcome; on the other hand, profound CD8+ T-cell lymphopenia, a high level of CD4+ and CD8+ T-cell activation and a high level of CD8+ T-cell senescence are associated with a higher mortality outcome. Furthermore, a cluster of patient was characterized by high B-cell responses with an extremely high level of plasmablasts. Our study points out the prognostic value of lymphocyte parameters such as T-cell activation and senescence and strengthen the interest in treating the patients early in course of the disease with targeted immunomodulatory therapies based on the type of adaptive response of each patient.

International audience; Introduction: Fertility disorders in autoimmune diseases are well described. However, little is known about the presence of a humoral serum autoimmunity in case of infertility (antinuclear antibodies, ACAN or antiphospholipid, APL) without criteria of autoimmune disease.Methods: We studied the prevalence, associated factors, and efficacy of immunomodulatory therapy in patients with unexplained infertility. Two groups were created retrospectively among patients followed in medically assisted procreation (PMA) for infertility: a group with serum autoimmunity (AI+) (ACAN, APL or anti-thyroperoxidase antibodies) and a group without serum autoimmunity (HAVE-). Clinical, biological, and therapeutic data were collected.Results: The prevalence of autoimmunity was 33% among consultant patients. One hundred patients were seen in internal medicine consultation, 70 were included in the AI+ group and 30 in the AI- group. In the AI+ group, 76% had ACANs, 29% had anti-TPOs and 23% had APLs. There was a significant correlation between ACAN level and the presence of endometriosis (P=0.048). Immunomodulatory therapy was introduced for 68 of the 70 women in the AI+ group; pregnancy occurred in 28 patients (40%) during the treatment period, compared with 7 in the "AI-" group (23%), with a tendency to significance (P=0.09). In conclusion, there is an increased prevalence of serum autoimmunity in patients with fertility disorders, possibly with the efficacy of an immunomodulatory treatment to confront prospective therapeutic studies.

International audience; Bradykinin mediated angioedema (BK-AE) can be associated either with C1Inhibitor deficiency (hereditary and acquired forms), either with normal C1Inh (hereditary form and drug induced AE as angiotensin converting enzyme inhibitors…). In case of high clinical suspicion of BK-AE, C1Inh exploration must be done at first: C1Inh function and antigenemy as well as C4 concentration. C1Inh deficiency is significant if the tests are below 50 % of the normal values and controlled a second time. In case of C1Inh deficiency, you have to identify hereditary from acquired forms. C1q and anti-C1Inh antibody tests are useful for acquired BK-AE. SERPING1 gene screening must be done if a hereditary angioedema is suspected, even if there is no family context (de novo mutation 15 %). If a hereditary BK-AE with normal C1Inh is suspected, F12 and PLG gene screening is suitable.

Ficolins are recognition proteins of the lectin pathway of the complement system and also play an important role in innate immunity and in the maintenance of tissue homeostasis. They deserve special attention in the context of autoimmunity since they are involved in the uptake of dying cells. Because the monitoring of systemic lupus erythematosus (SLE) patients is particularly difficult, it is crucial to find new relevant serum biomarkers. The ability to detect autoantibodies in the patients' sera provides a diagnostic and prognostic advantage. We describe in this chapter quantitative enzyme linked immunosorbent assays (ELISA) to detect the presence of autoantibodies targeting ficolin-2 and ficolin-3 in human sera. Recombinant ficolins produced in a mammalian expression system are used as coating antigens. The described in-house ELISAs provide a valuable tool to efficiently quantify anti-ficolin autoantibodies in the sera of SLE patients.

Background Studies of normal bone marrow (BM) cell composition by flow cytometry are scarce. Presently, we aimed to quantify 14 cell subsets from infants to elderly patients. Methods Cell subsets in BM samples from 180 individuals without morphologically abnormal leukocytes were analyzed using a single combination of eight antibodies: CD3/CD10/CD38/CD19/CD36/CD16/CD34/CD45. Results By comparison with the Holdrinet score, we first validated the immature granulocyte/neutrophil (IGRA/N) ratio as a readily obtainable criterion of BM sample purity in 145 cases. Then, the 115 highly pure samples were selected (IGRA/N ≥ 1.2) and analyzed according to age group. CD34+ myeloblasts became progressively more infrequent with age: median 1.4% in infancy to 0.5% in the elderly. Neutrophils increased: 10.7% to 22.8%; all other myeloid subsets, IGRA, eosinophils, basophils and monocytes remained stable: respectively 40.3% to 46.7%, 2.0% to 2.8%, 0.2% to 0.3%, and 4.4% to 5.0% throughout life. Erythroblasts were lower in children (8.4% to 10.3%) than in adults (12.5% to 15.1%). For lymphoid cells, hematogones and transitional B-cells decreased: 15.5% to 0.6% and 3.6% to 0.1%, respectively; mature lymphocytes remained stable: B-cells: 1.4% to 2.8%, T-cells: 5.8% to 8.7%, and NK-cells: 0.7% to 1.4%. Plasma cells varied slightly: 0.1% to 0.5%. Differences of about 40% were seen in moderately pure (IGRA/N: 0.5 to 1.2) BM samples. Conclusion We thus provide the first values for 14 myeloid and lymphoid subsets characterizing BM cell composition in 5 age ranges. They should provide important information when screening patients for hematological disorders or abnormal bone marrow development. © 2018 International Clinical Cytometry Society.

Detection of circulating anti-GBM antibodies has a key role for the diagnosis of Goodpasture syndrome but immunoassays using purified or recombinant alpha3(IV)NC1 as antigen do not recognize all anti-GBM antibodies. We show that anti-GBM antibodies directed against epitopes in their native conformation or cryptic epitopes are detected by indirect immunofluorescence.

Rheumatoid arthritis (RA) is a debilitating disease, but patient management and treatment have been revolutionized since the advent of bDMARDs. However, about one third of RA patients do not respond to specific bDMARD treatment without clear identified reasons. Different bDMARDs must be tried until the right drug is found. Here, we sought to identify a predictive protein signature to stratify patient responsiveness to rituximab (RTX) among patients with an insufficient response to a first anti-TNFα treatment.Serum samples were collected at baseline before RTX initiation. A proteomics study comparing responders and nonresponders was conducted to identify and select potential predictive biomarkers whose concentration was measured by quantitative assays. Logistic regression was performed to determine the best biomarker combination to predict good or nonresponse to RTX (EULAR criteria after 6 months' treatment).Eleven biomarkers potentially discriminating between responders and nonresponders were selected following discovery proteomics. Quantitative immunoassays and univariate statistical analysis showed that fetuin-A and thyroxine binding globulin (TBG) presented a good capacity to discriminate between patient groups. A logistic regression analysis revealed that the combination of fetuin-A plus TBG could accurately predict a patient's responsiveness to RTX with an AUC of 0.86, sensitivity of 80%, and a specificity of 79%.In RA patients for whom a first anti-TNFα treatment has failed, the serum abundance of fetuin-A and TBG before initiating RTX treatment is an indicator for their response status at 6 months. ClinicalTrials.gov identifier: NCT01000441. Key Points • Proteomic analysis revealed 11 putative predictive biomarkers to discriminate rituximab responder vs. nonresponder RA patients. • Fetuin-A and TBG are significantly differentially expressed at baseline in rituximab responder vs. nonresponder RA patients. • Algorithm combining fetuin-A and TBG accurately predicts response to rituximab in RA patients with insufficient response to TNFi.

Background Bone marrow analysis by flow cytometry is part of the routine diagnosis of hematological disorders in medical laboratories. Differential leukocyte count and identification of abnormal cell subsets is currently performed through morphological examination on bone marrow smears by skilled cytologists. In this work, we propose a single 8-color tube for providing equivalent information, using flow cytometry. Methods 99 bone marrow samples were classified into 2 groups, (i) 51 samples, obtained from either healthy donors (n = 4) or patients with various diseases at diagnosis or during remission that did not present a hematological malignancy (n = 47), and (ii) 48 pathological samples with quantitative and/or qualitative abnormalities. A panel of eight antibodies-CD3-FITC/CD10-PE/CD38-PerCP-Cy5.5/CD19-PECy7/CD36-APC/CD16-APC-H7/CD34-BV421/CD45-V500-was tested to identify the main cell subsets at different stages of maturation using a FACSCanto-II analyzer. Results We first proposed a strategy of sequential gating leading to the identification of 14 leukocyte subsets, that is, erythroblasts, monocytes, B-lymphoid cells from hematogones to plasma-cells (5 subsets), T- and NK-cells, polymorphonuclear cells (neutrophils, eosinophils, and basophils), myeloblasts and other immature granular cells. This approach was validated by comparing flow cytometry and microscopic morphological examination, both in cases of normal and abnormal samples. Interestingly, cell identification, and numeration by flow cytometry was easy to perform and highly reproducible. Conclusion A very simple, rapid, and reproducible flow cytometric approach, using a combination of eight antibodies allows determination of the cellular composition of bone marrow with high precision. © 2016 International Clinical Cytometry Society.

International audience; Traditionally, IBD diagnosis is based on clinical, radiological, endoscopic, and histological criteria. Biomarkers are needed in cases of uncertain diagnosis, or to predict disease course and therapeutic response. No guideline recommends the detection of antibodies (including ASCA and ANCA) for diagnosis or prognosis of IBD to date. However, many recent data suggest the potential role of new serological markers (anti-glycan (ACCA, ALCA, AMCA, anti-L and anti-C), anti-GP2 and anti-GM-CSF Ab). This review focuses on clinical utility of these new serological markers in diagnosis, prognosis and therapeutic monitoring of IBD. Literature review of anti-glycan, anti-GP2 and anti-GM-CSF Ab and their impact on diagnosis, prognosis and prediction of therapeutic response was performed in PubMed/MEDLINE up to June 2014. Anti-glycan, anti-GP2 and anti-GM-CSF Ab are especially associated with CD and seem to be correlated with complicated disease phenotypes even if results differ between studies. Although anti-glycan Ab and anti-GP2 Ab have low sensitivity in diagnosis of IBD, they could identify a small number of CD patients not detected by other tests such as ASCA. Anti-glycan Abs are associated with a progression to a more severe disease course and a higher risk for IBD-related surgery. Anti-GP2 Ab could particularly contribute to better stratify cases of pouchitis. Anti-GM-CSF Ab seems to be correlated with disease activity and could help predict relapses. These new promising biomarkers could particularly be useful in stratification of patients according to disease phenotype and risk of complications. They could be a valuable aid in prediction of disease course and therapeutic response but more prospective studies are needed.

Introduction Le traitement des uveites chroniques non-infectieuses peut inclure l’utilisation d’anticorps monoclonaux anti-TNFα, dont l’adalimumab (ADA) et l’infliximab (IFX). Grâce a l’utilisation de tests commerciaux ELISA, la detection d’anticorps anti-adalimumab (AAA) ou anti-infliximab (AAI) et le dosage de la concentration sanguine d’anti-TNFα peuvent etre realises en pratique clinique courante. L’objectif de notre etude est d’evaluer l’interet de ces dosages, dans la prise en charge des patients presentant une uveite chronique non infectieuse. Patients et methodes Une etude observationnelle monocentrique retrospective a ete realisee en 2017 au Centre Hospitalier Universitaire Grenoble Alpes. Les patients presentant une uveite chronique non infectieuse, traites par adalimumab ou par infliximab, et ayant eu un dosage du taux plasmatique du medicament et une recherche d’anticorps anti-traitement (AAA ou AAI), ont ete inclus. Le dosage du medicament et la recherche d’anticorps induits ont ete realises par technique ELISA (Theradiag®). La reponse ophtalmologique de l’uveite (totale, partielle ou absente) etait determinee a chaque dosage. Cette reponse etait definie, par rapport a l’etat clinique a l’instauration du traitement, selon un critere clinique composite ophtalmologique base sur la presence d’inflammation dans la chambre anterieure ou le vitree, d’un œdeme maculaire cystoide ou d’anomalies a l’angiographie. Resultats Vingt et un patients suivis pour une uveite chronique non infectieuse sous anti-TNFα, dont 12 traites par adalimumab et 9 par infliximab, ont beneficie d’au moins une recherche d’anticorps anti-medicament et d’un dosage sanguin d’anti-TNFα. Trois dosages ont ete realises chez 7 patients, 2 dosages chez 3 patients et enfin, 1 dosage chez 11 patients. Trois patients ont developpe des anticorps. Ils etaient transitoires pour un patient traite par adalimumab. Ce patient poursuit actuellement le traitement sans perte d’efficacite. Ils etaient stables pour deux patients (un traite par IFX et un par ADA) qui ont arrete le traitement pour inefficacite clinique. Alors qu’une reponse clinique totale et partielle etait retrouvee chez 60,5 % des patients en absence d’anticorps, une absence de reponse clinique etait systematiquement retrouvee en presence d’AAA ou d’AAI permanents. En presence d’AAA ou d’AAI, la concentration sanguine d’anti-TNF α etait plus faible : 3,2 ± 5,4 μg/mL [0,1–9,4] vs 9,8 ± 5,0 μg/mL [0,1–16] (p = 0,032). Neanmoins, aucune association entre concentration sanguine et efficacite clinique n’a pu etre mise en evidence. Conclusion Aucune relation, entre l’apparition d’anticorps anti-medicament et l’efficacite clinique sur l’uveite chronique non infectieuse, n’a pu etre mise en evidence. Neanmoins, tous les patients ayant developpes des AAA ou AAI stables ont arrete le traitement pour absence d’efficacite clinique. L’apparition d’anticorps anti-medicament est associee a une diminution de la concentration sanguine de l’anti-TNFα. Une etude prospective semble interessante a mener, pour montrer l’interet des dosages sanguin d’anti-TNFα et d’anticorps anti-medicament dans les uveites chroniques non-infectieuses.

Introduction Les auto-anticorps (auto-Ac) anti-SSA et anti-SSB sont presents dans plusieurs pathologies dysimmunitaires, parmi lesquelles le syndrome de Goujerot Sjogren (SGS), le lupus erythemateux dissemine (LED) ou la Polyarthrite Rhumatoide (PR). Si la sensibilite et la specificite de ces auto-Ac sont actuellement bien decrites, nous avons voulu determiner les etiologies associees a la presence de ces auto-Ac. Patients et methodes Nous avons mene une etude retrospective, observationnelle, monocentrique au CHU de Grenoble-Alpes. Tous les patients ayant beneficie d’une recherche positive d’auto-Ac anti-SSA et/ou anti-SSB entre janvier 2013 et decembre 2015 ont ete inclus ; seuls les premiers dosages pour chaque patient ont ete retenus. Pour chaque dossier, etaient relevees les donnees demographiques, cliniques et biologiques. Resultats Deux cent vingt-neuf recherches positives d’auto-Ac anti-SSA et/ou SSB ont ete recensees sur la periode. Parmi elles, 100 patients avaient beneficie de cette recherche pour la premiere fois au CHUGA. Une recherche d’auto-Ac anti-nucleaires (ACAN) etait positive en immunofluorescence pour 97 patients. La presence d’auto-Ac anti-SSA60, -SSA52 et -SSB etait isolee respectivement dans 96 %, 59 % et 45 % des cas. Les diagnostics principalement associes a un dosage positif etaient : SGS (42 %), LED (27 %), syndrome des anti-phospholipides isole (7 %), thyroidite auto-immune (6 %), PR (5 %), pseudo polyarthrite rhizomelique (2 %), sclerodermie (3 %) ( Tableau 1 ). Pour 20 patients, 2 diagnostics etaient associes, notamment LES et SAPL (6 %), LES et SGS (5 %). Par ailleurs, on notait 1 cas de vascularite urticarienne hypo-complementemique, et 9 (9 %) tableaux cliniques evocateurs de pathologie dysimmunitaire sans diagnostic retenu. Les symptomes rapportes (pour 83 patients) etaient : syndrome sec (43 %), arthralgie (46 %), manifestation cutanee (19 %) et syndrome de Raynaud (17 %). Conclusion Cette etude monocentrique retrospective sur 100 patients presentant un premier dosage positif d’auto-Ac anti-SSA et/ou SSB, confirme la part importante de SGS associee, mais montre aussi les prevalences non negligeables de LED et de SAPL.

To cite this article: Vitrat-Hincky V, Gompel A, Dumestre-Perard C, Boccon-Gibod I, Drouet C, Cesbron JY, Lunardi J, Massot C, Bouillet L. Type III hereditary angio-oedema: clinical and biological features in a French cohort. Allergy 2010; 65: 1331–1336. Abstract Background: Hereditary angio-oedema (HAE) has been associated with C1inhibitor deficiency. The first cases of type III HAE were described in patients with normal C1Inh antigenic protein level and function and normal C4 levels in 2000. This finding has been reported mostly in women with a family history and may be influenced by exogenous oestrogen exposure. Objectives: The purpose of this article is to describe the clinical, biological and genetic characteristics of a French population suffering from type III HAE. Patients and Methods: We conducted a retrospective analysis of angio-oedema (AE) cases seen in the National Reference Centre of AE between 2000 and 2009. Results: We found 26 patients (from 15 unrelated families) with type III HAE. All but four were women and presented with typical AE attacks, exacerbated by pregnancy or oral contraceptives containing oestrogens (OC). We also found that 54.5% of women were worsened with oestrogen and 23% were oestrogen dependent. All patients improved on long-term prophylactic tranexamic acid treatment; some acute attacks improved with C1Inh concentrate infusion. All of the patients had normal C1Inh and C4 levels. C1Inh function was also normal, except in women receiving OC or during a pregnancy: transient, moderately low levels (32–74% of the normal range) were found in 18 patients tested (67%). No SERPING1 gene mutation was found. Six patients from three unrelated families were heterozygous for an F12 gene variant. Conclusion: Diagnosis of type III HAE should be based on clinical (typical attacks, often hormonally influenced), laboratory (normal C1Inh antigenic protein) and genetic (F12 gene mutation) evidence.

Ficolins are oligomeric innate immune recognition proteins consisting of a collagen-like region and a fibrinogen-like recognition domain that bind to pathogen- and apoptotic cell-associated molecular patterns. To investigate their carbohydrate binding specificities, serum-derived L-ficolin and recombinant H- and M-ficolins were fluorescently labeled, and their carbohydrate binding ability was analyzed by glycan array screening. L-ficolin preferentially recognized disulfated N-acetyllactosamine and tri- and tetrasaccharides containing terminal galactose or N-acetylglucosamine. Binding was sensitive to the position and orientation of the bond between N-acetyllactosamine and the adjacent carbohydrate. No significant binding of H-ficolin to any of the 377 glycans probed could be detected, providing further evidence for its poor lectin activity. M-ficolin bound preferentially to 9-O-acetylated 2-6-linked sialic acid derivatives and to various glycans containing sialic acid engaged in a 2-3 linkage. To further investigate the structural basis of sialic acid recognition by M-ficolin, point mutants were produced in which three residues of the fibrinogen domain were replaced by their counterparts in L-ficolin. Mutations G221F and A256V inhibited binding to the 9-O-acetylated sialic acid derivatives, whereas Y271F abolished interaction with all sialic acid-containing glycans. The crystal structure of the Y271F mutant fibrinogen domain was solved, showing that the mutation does not alter the structure of the ligand binding pocket. These analyses reveal novel ficolin ligands such as sulfated N-acetyllactosamine (L-ficolin) and gangliosides (M-ficolin) and provide precise insights into the sialic acid binding specificity of M-ficolin, emphasizing the essential role of Tyr271 in this respect.

Mice defective for C1q complement factor show enhanced resistance to peripheral prion inoculation, and previous work demonstrated a direct interaction between C1q and conformationally modified PrP. However, the nature and physiological consequences of this interaction remain uncharacterized. PrP amino acids 141-159 has been identified as a potential C1q binding site; we show, by both surface plasmon resonance (SPR) spectroscopy and ELISA, that C1q and its globular region bind to PrP mutagenized in the region of interest with comparable efficiency to that of wild-type protein. To test PrP's ability to activate complement, soluble oligomers of the PrP constructs were made. Only PrP and mutagenized PrP oligomers activate the classical complement cascade while PrP monomer and the C-terminal domain, both in oligomeric and in monomeric form, failed to induce activation. This suggests that a conformational change in PrP, which occurs both when PrP is bound to an SPR sensor chip and when it undergoes oligomerization, is requisite for PrP/C1q interaction and activation of the complement cascade. We propose that C1q may act as a natural sensor for prions, leading to activation of the classical complement cascade, which could result in local inflammation and subsequent recruitment of the immune cells that prions initially infect.

The aetiology of angio-oedema (AE) is difficult to determine; however, it is essential in emergency situations when two major contexts may be present: mast cell-mediated AE and bradykinin-mediated AE. Different forms of AE are currently distinguished based on clinical criteria (spontaneous duration of the attack, presence of concomitant or late-appearing superficial urticaria, history of atopy, and others), but specific biomarkers could improve patient management.In this prospective study, potential biomarkers have been identified, and their statistical characteristics were examined.Samples were taken on day 0 (D0) and D7 for 3 patient groups (n = 11 each): bradykinin-mediated AE [peripheral site of attack, ear, nose, throat (ENT), and abdominal involvement], mast cell-mediated AE, and non-bradykinin-mediated abdominal pain.Assay of the potential biomarkers revealed no significant differences in C1 inhibitor and C4 levels. In contrast, D-dimer levels peaked during bradykinin-mediated AE attacks (median 2.2 mg/l at D0 vs. 0.52 mg/l at D7; p10-3) as well as during mast cell-mediated AE attacks (1.97 vs. 0.65 mg/l; p = 0.04) and were high in bradykinin-mediated AE compared to the control group (0.69 mg/l; p = 0.01). A threshold value of 0.62 mg/l was found to have a negative predictive value of 100% for bradykinin-mediated AE compared to other causes of abdominal pain (group 3). Circulating VE-cadherin levels were also increased during an attack (1,990 at D0 vs. 1,566 ng/ml at D7; p = 0.01), but could not distinguish between bradykinin-mediated and mast cell-mediated AE, like D-dimers.Exploration of changes in fibrinolysis-related markers (particularly D-dimers) is thus promising for the diagnosis of AE attacks in difficult-to-diagnose abdominal forms, although it was not able to differentiate between bradykinin and mast cell-mediated AE.

International audience; Most patients with idiopathic membranous nephropathy (IMN) have IgG4 autoantibodies against phospholipase A2 receptor (PLA2R). C3 and C5b-9 are found in immune deposits of IMN kidney biopsy specimens, but the pathway of complement activation in IMN remains elusive. We report the case of a patient who developed IMN with intense staining for PLA2R, IgG4, C3, C5b-9, factor B, and properdin and very weak staining for C1q, C4d, and IgG1. Measurement of mannan binding lectin (MBL) antigenic level and activity revealed MBL deficiency. Genotyping revealed a heterozygous (A/C) polymorphism in codon 57 of MBL2 exon 1 associated with homozygous and heterozygous variations in the promoter region at -550 (L/L) and -221 (X/Y), respectively, suggesting that the patient harbored the LXA/LYC haplotypes linked to MBL deficiency. Genetic sequencing in 77 consecutive patients with IMN identified four patients with MBL2 promoter and coding region variations associated with MBL deficiency and the same complement pattern in immune deposits as the index patient. In contrast, patients with wild-type MBL2 had immune deposits with intense Cd4 staining. Thus, IMN can develop in patients with complete MBL deficiency, with complement activated mainly by the alternative pathway, whereas the lectin pathway is also activated in those with wild-type MBL2.

The objective of the present work was to set up a routine test adapted to screening for antiendothelial cell antibodies (AECAs) in serum samples with minimal interference from antinuclear antibodies (ANAs) or rheumatoid factors (RFs). We compared the titers of AECAs titrated following two enzyme-linked immunosorbent assays (ELISAs): (i) an ELISA with ethanol-fixed EA.hy926 monolayers as the antigenic substrate and (ii) an ELISA with nucleus-depleted lysates prepared from EA.hy926 cells and normalized for protein (1.0 to 1.7 mg/ml) and DNA (≤0.1 μg/ml) contents as a surrogate substrate (postnuclear supernatant ELISA [PNS-ELISA]). The AECA titers in 51 serum samples, including 28 samples containing ANAs, were compared. A significantly positive correlation ( r = 0.77; P < 0.001) between the two series was shown only for the ANA-negative serum samples. Conversely, ANAs or RFs in samples were shown not to interfere in tests for AECAs by the PNS-ELISA. AECAs recognize their antigenic targets in postnuclear supernatants, which is representative of the endothelial antigenic content, with improvement of the reliability of the assay, a prerequisite to application of the assay for their evaluation in clinical practice.

International audience; To identify patients with autoimmune diseases who are at high risk of developing vascular cell dysfunction, early biomarkers must be identified. This study was designed to detect and characterize circulating autoantibodies to VE-cadherin (AAVEs) in patients with early-stage autoimmune diseases. An enzyme-linked immunosorbent assay (ELISA) was developed to capture autoantibodies, using a recombinant human VE-cadherin fragment covering the extracellular domains as a target antigen. AAVEs specificity for the target antigen was confirmed by western blotting. Basal AAVEs levels were determined for healthy donors (n=75). Sera from patients (n=100) with various autoimmune diseases, including rheumatoid arthritis (n=23), systemic lupus erythematosus (SLE, n=31), systemic sclerosis (n=30), and Behçet's disease (BD, n=16) were also tested. Levels of AAVEs were significantly higher in rheumatoid arthritis (P

Introduction Les angiœdemes acquis histaminiques idiopathiques (AOA-HI) sont une cause frequente d’angiœdemes (AO) sans urticaire [1] , [2] . Il s’agit d’une maladie mediee par les mastocytes, qui est decrite comme une forme clinique particuliere de l’urticaire chronique. Peu de donnees sont disponibles concernant cette variete d’AO. Les objectifs de cette etude sont de decrire les caracteristiques cliniques et epidemiologiques des patients souffrant d’AOA-HI. Methodes De 2014 a 2015, 534 patients ont ete vus au centre national de reference sur les angiœdemes (CREAK). Parmi eux, nous avons identifie 31 patients atteints d’AOA-HI, que nous avons inclus dans cette etude. Resultats Parmi les 31 patients souffrant d’AOA-HI, un ratio de 15 hommes pour 16 femmes a ete retrouve. Le delai diagnostique moyen etait de 6,3 ans. La duree moyenne des crises etait de 28,1 heures. La crise d’AO interessait la sphere ORL dans 54,8 % des cas. Une localisation linguale a ete trouvee dans 29 % des cas. Les hommes avaient un risque plus important que les femmes d’avoir une localisation ORL de leurs crises. Aucune intubation ni deces n’ont ete releves. La dose d’antihistaminiques necessaire pour controler les symptomes etait d’un comprime par jour pour 51,6 % des patients, 2 comprimes par jour pour 32 %, et 3 a 4 comprimes pour 16,1 % des patients. Conclusion Les AOA-HI sont caracterises par un retard diagnostique important, une atteinte frequente des voies respiratoires superieures pendant les crises, mais aucune atteinte severe avec mise en jeu du pronostic vital n’a ete rapportee. Comme au cours de l’urticaire chronique, un traitement antihistaminique de fond jusqu’a quatre fois la dose de l’AMM peut etre necessaire pour controler les symptomes.

International audience; Phagocytes play a central role in immune defense. Their dysfunction predisposes to infections. This study determined the expression level of nine receptors involved in Aspergillus immune response as well as the values of phagocytosis and production of radical oxygen species after Aspergillus stimulation, in a healthy adult population. The expression values of the CD11b, CD11c, CD14, CD18, CD35, CD181, CD182, CD282 and CD284 receptors on peripheral human monocytes and granulocytes was established. A heterogenous expression of the CD282 on granulocytes was observed as CD181, CD182 and CD284 on monocytes. Similarly, we observed considerable variation in the expression of these receptors over time. Only CD282 on granulocytes varied with sex. No variation with age was observed. Adherence of Aspergillus conidia to phagocytes was dependent of individual, sex, age and time. A better characterization of these innate immunity parameters is necessary to develop in the future an immunologic surveillance strategy for transplant recipients.

Innate immunity is the major host defense against invasive aspergillosis. To determine whether the collectin mannan-binding lectin (MBL) is involved in the initial protective immunity through complement activation against opportunistic fungal infections caused by Aspergillus, we performed in vitro studies on 29 different strains of Aspergillus conidia from five different species. Incubation of Aspergillus conidia in human normal serum leads to activation of the alternative pathway, whereas neither the classical nor the lectin pathways through C4 and C2 cleavage are activated. Complement response to conidia was investigated using a MBL-deficient serum and reconstitution experiments were conducted with MBL/MASPs complexes. We found that MBL can directly support C3 activation by a C2 bypass mechanism. Finally, a stronger activation of the alternative pathway was observed for the clinical strains isolated from patients with invasive aspergillosis, compared with the environmental strains.

The complement system is a major humoral portion of the innate immune system, playing a significant role in host defence against microorganisms. The biological importance of this system is underlined by the fact that at least three different pathways for its activation exist, the classical, the MBL and the alternative pathway. To elucidate the involvement of the classical and/or the MBL pathway during bacterial septicemia, 32 patients with gram-positive and 30 patients with gram-negative bacterial infections were investigated. In patients with gram-positive bacteria, a significant consumption of C1q (p=0.005) but not of mannose-binding lectin (MBL) (p=0.2) was found during the acute phase of infection. In contrast, in patients with gram-negative bacterial infections, a significant reduction of MBL (p=0.002) and only a moderate, less significant reduction of C1q (p=0.03) were observed. As a model for the binding of MBL to gram-negative bacteria, Salmonella strains with defined mutations in their lipopolysaccharide (LPS) structure were used. The comparison of the binding of MBL to these Salmonella strains with that of the corresponding isolated LPS forms bound to microtiter plates revealed a similar binding pattern, supporting the interpretation that LPS on the surface of gram-negative bacteria is the major acceptor molecule for MBL on these bacteria, which according to our results obviously also takes place during gram-negative bacterial septicaemia. Furthermore, we were able to demonstrate that MBL bound to LPS was able to initiate activation of the complement cascade as measured by the occurrence of the cleavage product C4c.

Human mannan binding lectin (MBL) is a member of the collectins, a group of proteins that contain a dual structure with a lectin and a collagenous moieties. The collectins are considered as major actors of innate immunity. We report the presence of low molecular weight intracellular MBL forms in human hepatocytic cell lysates, with binding capacities associated to its lectin and/or its collagen moiety. Competition with D-mannose and with antibodies directed against the lectin binding site of MBL indicate that the 60 kDa form represents an intracellular association of MBL through its lectin moiety. The effects of collagenase or MBL associated serine proteases (MASPs) from a MBL deficient plasma, gave evidence that the 60 KDa form contains also collagen and suggested the binding of a ligand to this collagen part. These results show that this intracellular form of MBL shares binding properties with circulating MBL. The binding potential of the lectin and the collagenous parts of precursor forms of intracellular MBL may suggest that they behave as molecular chaperone. The complexity of MBL structure and functions deserves further investigation on other intracellular forms of MBL.

The mannan binding lectin (MBL) plays a major role in innate immunity through its ability to activate complement upon binding to carbohydrate arrays on the surface of various microorganisms. The question of a possible association of the MBL structural gene polymorphism and the oligomeric state of MBL was poorly documented. For these reasons, it appears difficult to evaluate MBL in blood patients on the only basis of protein contents, even in combination with MBL genotyping. This study reports a method to calculate a specific activity for circulating MBL, that relies on: (i) the availability of purified MBL; and (ii) a simplified MBL activity assay based on complement activation. The three-step MBL purification from human plasma reported here is characterized by a highly purified MBL, that occurs in two different oligomeric forms. The results on the specific activity of these forms show that the higher oligomeric forms of MBL have the ability to induce C4 cleavage more efficiently than the corresponding lower oligomers. The usefulness of this approach is illustrated by its potential interest in the biological exploration of certain pathology, for example in the follow-up of chronic hepatitis C. Further investigation is needed to establish whether MBL specific activity (MBLsa) is correlated to the polymorphic state of the molecule. The relative simplicity of the test described here allows better investigation on the relationship between MBL biological activity and its genotype.

SummaryThe overall role of complement in the host–pathogen relationship is now well understood. However, its involvement at a chronic stage of infection, such as chronic hepatitis C, is less well documented. Here, results are reported which point to the use of specific C4 monitoring in the follow-up of HCV patients. This study concerns 66 patients with chronic HCV infection, treated with interferon alpha 2b alone or with interferon alpha 2b + ribavirin, and 50 healthy adults as controls. Complement blood tests were performed to measure C1q, C3, C4, mannan binding lectin (MBL), C1s-C1 inhibitor complexes, total (CH50) and C4 (C4H) haemolytic activity; specific C4 activity was taken as the C4H/C4 protein ratio. Rheumatoid factor (RF) levels were also measured. A significant reduction in CH50 and specific C4 activity in HCV patients, compared with the healthy controls, was observed before the onset of treatment; the other parameters were not affected and no C1s-C1 inhibitor complexes were detected. At the same time, a significant reduction in specific C4 activity was observed in relapsers compared with sustained responders. These results point to a potential predictive function of C4 specific activity to monitor the response to therapy. Restoration of specific C4 activity at 6 months was better in responders than in non-responders. Complement activation in chronic hepatitis C does not seem to involve the C1 stage of the classical pathway. A negative correlation between specific C4 activity and RF titres suggests a possible involvement of RF in C4 activation, via the lectin pathway. Specific C4 monitoring appears to be a valuable tool for the follow-up of chronic hepatitis C treatment, together with the other conventional investigations.

Innate immunity is the major host defense against invasive aspergillosis. To determine whether the collectin mannan-binding lectin (MBL) is involved in the initial protective immunity through complement activation against opportunistic fungal infections caused by Aspergillus, we performed in vitro studies on 29 different strains of Aspergillus conidia from five different species. Incubation of Aspergillus conidia in human normal serum leads to activation of the alternative pathway, whereas neither the classical nor the lectin pathways through C4 and C2 cleavage are activated. Complement response to conidia was investigated using a MBL-deficient serum and reconstitution experiments were conducted with MBL/MASPs complexes. We found that MBL can directly support C3 activation by a C2 bypass mechanism. Finally, a stronger activation of the alternative pathway was observed for the clinical strains isolated from patients with invasive aspergillosis, compared with the environmental strains.