Chantal Dumestre-Pérard

Carbohydrate Recognition Properties of Human Ficolins: GLYCAN ARRAY SCREENING REVEALS THE SIALIC ACID BINDING SPECIFICITY OF M-FICOLIN*

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.

Type III hereditary angio-oedema: clinical and biological features in a French cohort

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.

Residue Lys57 in the Collagen-Like Region of Human L-Ficolin and Its Counterpart Lys47 in H-Ficolin Play a Key Role in the Interaction with the Mannan-Binding Lectin-Associated Serine Proteases and the Collectin Receptor Calreticulin

L- and H-ficolins are serum oligomeric defense proteins consisting of a collagen-like region and a fibrinogen-like recognition domain that bind to pathogen- and apoptotic cell-associated molecular patterns. They share with mannan-binding lectin (MBL) the ability to associate with MBL-associated serine proteases (MASP)-1, -2, -3, and protein MAp19 and to trigger the lectin complement pathway through MASP-2 activation. Recent studies have revealed the essential role of Lys55 in the collagenous region of MBL in the interaction with the MASPs and calreticulin (CRT). To test the possible involvement of the homologous residues Lys57 of L-ficolin and Lys47 of H-ficolin, point mutants of both proteins were produced in which these residues were mutated to Ala, Glu, or Arg. The resulting mutants exhibited oligomerization patterns and ligand binding properties similar to those of their wild-type counterparts. In contrast, all three mutations strongly inhibited the interaction of L- and H-ficolins with MAp19 and MASP-2 and impaired the ability of each ficolin to trigger the lectin pathway. In the case of MASP-1 and MASP-3, replacement of the target Lys residues by Ala or Glu abolished interaction, whereas the Lys to Arg mutations had only slight inhibitory effects. Likewise, binding of each ficolin to CRT was inhibited by mutation of Lys to Ala or Glu, but not to Arg. In conclusion, residues Lys57 of L-ficolin and Lys47 of H-ficolin are key components of the interaction with the MASPs and CRT, providing strong indication that MBL and the ficolins share homologous binding sites for both types of proteins.

BRADYKININ RECEPTOR 2 ANTAGONIST (ICATIBANT) FOR HEREDITARY ANGIOEDEMA TYPE III ATTACKS

In 2000, a new type of hereditary angioedema (HAE) without C1 esterase inhibitor (C1-INH) deficiency, HAE type III, was reported.1,2 It occurred especially in women, and the diagnosis was based on recurrent attacks of subcutaneous or submucosal edema, no chronic relapsing urticaria, familial history, and normal C1INH protein and activity levels. Recent findings3 suggest that bradykinin may play a role in the pathogenesis of angioedema. The morbidity and mortality of HAE are related to abdominal attacks and laryngeal edema. Symptoms seem to be induced or worsened by estrogen.4 In these patients, C1-INH function is normal, and a missense gain-of-function mutation in the F12 gene can be found.5 Treatments for HAE type III are not established, but tranexamic acid seems to be effective for prophylaxis of symptoms. Until recently, the main shortterm treatment for severe HAE attacks was C1-INH concentrate.4 A new therapeutic agent, icatibant, a bradykinin B2 receptor antagonist, is effective for the treatment of HAE with C1-INH deficiency. The efficacy and safety of icatibant in patients with HAE (type I or II) presenting with moderate to severe cutaneous or abdominal attacks were tested in 2 double-blind, randomized, multicenter, phase 3 trials, FAST (For Angioedema Subcutaneous Treatment) 1 and 2.6 We report treatment outcomes in 3 women who fulfilled the diagnosis criteria of HAE type III (Table 1). Patients presented with recurrent severe abdominal attacks, edema of the face, and a family history of angioedema. The C1-INH function was moderately low when the patients took an estrogen-containing contraceptive pill or during pregnancy but was normal after it was discontinued or after delivery. We previously described these data in another patient with HAE type III.7 Levels of C1-INH protein and C4 were normal. All 3 patients had no mutation in the SERPING1 gene. Patient 1 had an identified mutation in the F12 gene. Patient 1 received 1 subcutaneous injection of icatibant, 30 mg, for a severe abdominal attack. Symptom improvement began 30 minutes later, with complete resolution of symptoms by 1 hour after icatibant administration. Patient 2 presented with an unusual severe abdominal attack combined with facial edema. An increase in her dose of tranexamic acid did not improve symptoms. She received an injection of icatibant. Symptoms were relieved within 2 hours. Patient 3 was treated for a severe abdominal attack. After the first injection of icatibant, relief of symptoms occurred within 30 minutes. Symptom recurrence after 6 hours necessitated a second injection of icatibant, which resulted in rapid alleviation of symptoms. For all the patients, icatibant administration was followed by a transient local erythematic reaction at the site of injection. No other adverse reactions to icatibant were noted in the 3 patients. We therefore conclude that icatibant is a safe and effective symptomatic treatment for severe attacks in patients with HAE type III.

Evaluation and clinical interest of mannan binding lectin function in human plasma.

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.

Detection of Antiendothelial Cell Antibodies by an Enzyme-Linked Immunosorbent Assay Using Antigens from Cell Lysate: Minimal Interference with Antinuclear Antibodies and Rheumatoid Factors

ABSTRACT 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.

Phospholipase A2 Receptor–Related Membranous Nephropathy and Mannan-Binding Lectin Deficiency

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.

Unfolded protein response gene GADD34 is overexpressed in rheumatoid arthritis and related to the presence of circulating anti-citrullinated protein antibodies.

Abstract Growth arrest and DNA damage-inducible gene 34 (GADD34) is an inducible cofactor of protein phosphatase 1, which has an important role in the unfolded protein response. GADD34 has been shown to be necessary for type I interferon and proinflammatory cytokine production in response to viral infection in murine models. We investigate the expression of GADD34 in rheumatoid arthritis (RA), in which proinflammatory cytokines have an important pathogenic role. The objective of this study was to evaluate the potential of GADD34 expression as a biomarker in RA patients. We report a case–control study on GADD34 gene expression in peripheral blood mononuclear cells of patients (n = 75) with RA and age- and sex-matched healthy controls (n = 25). The study was approved by the relevant local ethics committees. GADD34 gene expression level in peripheral blood mononuclear cells was measured by quantitative PCR and analyzed with Mann–Whitney test. The relation between GADD34 gene overexpression and clinical or biological characteristics was analyzed with univariate and multivariate analysis. GADD34 gene expression was significantly higher in RA patients compared with healthy controls (p ≤ 0.001). Interestingly, GADD34 overexpression in PBMC of patients was related to the presence of circulating anti-citrullinated protein antibodies (p = 0.030). Data of this study strengthen the evidence of an unfolded protein response during the course of RA and provide an insight of the potential interest in GADD34 as a relevant marker for RA.

One tube with eight antibodies for 14-part bone marrow leukocyte differential using flow cytometry

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.

Association between the Presence of Autoantibodies Targeting Ficolin-3 and Active Nephritis in Patients with Systemic Lupus Erythematosus

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by the production of multiple autoantibodies. Antibodies against Ficolin-3 were previously identified in the sera of some SLE patients, but their prevalence and significance have not been yet investigated. The aims of this study were to determine the prevalence of anti-ficolin-3 antibodies among SLE patients and to investigate their potential as diagnostic and/or prognostic biomarkers in SLE. In this retrospective study, sera from SLE patients (n = 165) were selected from a preexisting declared biological collection. Samples from healthy controls (n = 48) were matched with SLE sera. Disease activity was determined according to the SLEDAI score. Anti-ficolin-3, anti-dsDNA and anti-C1q antibodies levels were measured in sera by ELISA. First, a highly significant difference was found in the anti-ficolin-3 levels between SLE patients and healthy subjects. Anti-ficolin-3 antibodies were detected as positive in 56 of 165 (34%) SLE patients. The titer of anti-ficolin-3 antibodies was correlated with the SLEDAI score (r = 0.38, p<0.0001). The presence of anti-ficolin-3 antibodies was associated with anti-C1q and anti-dsDNA antibodies. Regarding associations with clinical manifestations, the presence of active lupus nephritis was significantly associated with the presence of anti-ficolin-3 antibodies (p≤0.001). This association with renal involvement was higher with anti-ficolin-3 or anti-C1q antibodies than with other auto-antibodies. Interestingly, the combination of anti-ficolin-3 and anti-C1q antibodies demonstrated higher specificity than any other serological biomarker. These results suggest that anti-ficolin-3 antibodies could be useful for the diagnosis of active nephritis in SLE patients.