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Introduction La glomerulonephrite a depots de C3 (GC3) est associee a la presence d’un auto-anticorps stabilisant la C3 convertase alterne (C3bBb), le C3 Nephritic Factor (C3NeF), conduisant a une activation incontrolee du complement. Cependant, sa technique de detection, le test hemolytique (TH), n’est pas standardisee et reservee aux laboratoires specialises. Nous avons donc developpe la recherche de C3NeF par Luminex®. Methodes La capacite de fixation et de stabilisation d’une C3 convertase alterne, formee sur bille, par des immunoglobulines G (IgG) de patients atteints de GC3 a ete etudiee. Ces resultats ont ete confrontes a ceux du TH. Les anticorps anti-C3b ont ete egalement recherches. La C3Bb est formee avec du facteur B et D sur des billes recouvertes de C3b. Les IgG purifiees de patients sont ensuite ajoutees, puis la fixation des IgG anti-C3bBb (ou anti-C3b sur billes seules) est quantifiee par un anticorps anti-IgG couple a la phycoerythrine (PE). La stabilisation de la C3 convertase alterne est revelee par un anticorps anti-Bb biotinyle, melange a la streptavidine-PE. Le seuil de positivite des tests (moyenne + 2 ecarts-types) est determine a partir des IgG de 30 donneurs sains et exprime en intensite de fluorescence moyenne (MFI). Resultats obtenus ou attendus Trente deux patients positifs en TH ont ete testes. 21/24 etaient positifs en stabilisation (p = 0,62 versus TH, kappa = 0,82) et 24/29 en fixation de la C3bBb (p = 1 versus TH, kappa = 0,72). 15/31 patients presentaient des anticorps anti-C3b. Trois patients negatifs en TH presentant une GC3 etaient positifs en IgG anti-C3bBb, suggerant une meilleure sensibilite du Luminex®. La MFI de l’IgG anti-C3bBb etait inversement correlee au dosage plasmatique de C3 (p = 0,0001). Conclusion Cette technique, simple de mise en œuvre, presente une bonne correlation avec le TH, et permettrait une meilleure detection des C3NeF et des anticorps anti-C3b. Ce test pourrait devenir l’outil de reference pour le diagnostic et le suivi des GC3.

International audience; In C3 glomerulopathy (C3G), the alternative pathway of complement is frequently overactivated by autoantibodies that stabilize the C3 convertase C3bBb. Anti-C3b and anti-factor B (anti-FB) IgG have been reported in three patients with C3G. We screened a cohort of 141 patients with C3G and Ig-associated membranoproliferative GN (Ig-MPGN) for anti-FB and anti-C3b autoantibodies using ELISA. We identified seven patients with anti-FB IgG, three patients with anti-C3b IgG, and five patients with anti-FB and anti-C3b IgG. Of these 15 patients, ten were diagnosed with Ig-MPGN. Among those patients with available data, 92% had a nephrotic syndrome, 64% had AKI, and 67% had a documented infection. Patients negative for anti-C3b and anti-FB IgG had much lower rates of infection (17 [25%] patients with C3G and one [10%] patient with Ig-MPGN). After 48 months, four of 15 (26%) positive patients had developed ESRD or died. All 15 patients had high plasma Bb levels, six (40%) patients had low levels of C3, and nine (60%) patients had high levels of soluble C5b9. In vitro, IgG purified from patients with anti-FB Abs selectively enhanced C3 convertase activity; IgG from patients with anti-C3b/anti-FB Abs enhanced C3 and C5 cleavage. IgG from patients with anti-C3b Abs stabilized C3bBb and perturbed C3b binding to complement receptor 1 but did not perturb binding to factor H. In conclusion, the prevalence of anti-C3b/anti-FB Abs and alternative pathway activation is similar in Ig-MPGN and C3G, suggesting similar pathogenic mechanisms. Identification of the underlying defect in Ig-MPGN could lead to improved treatment.

The innate immune complement system is a powerful defense cascade against pathogens, but can induce host tissue damage when overactivated. In pathological conditions, mainly but not restricted to renal diseases, such as lupus nephritis, atypical hemolytic uremic syndrome, and C3 glomerulopathies, complement is overactivated or dysregulated by autoantibodies directed against its components and regulators. Among the key autoantibody targets are the initiator of the classical complement pathway C1q, the alternative pathway regulator Factor H, the components of the alternative pathway C3 convertase complex C3 and Factor B and the convertase complex itself. This methodological article describes our experience with a method for detection of anti-complement autoantibodies in real time using surface plasmon resonance-based technology. It allows label-free evaluation of the binding efficacy and stability of the formed antigen-antibody complexes.

The complement system represents a major component of our innate immune defense. Although the physiological contribution of the complement system is beneficial, it can cause tissue damage when inappropriately activated or when it is a target of an autoantibody response. Autoantibodies directed against a variety of individual complement components, convertases, regulators and receptors have been described. For several autoantibodies the functional consequences are well documented and clear associations exist with clinical presentation, whereas for other autoantibodies targeting complement components this relation is currently insufficiently clear. Several anti-complement autoantibodies can also be detected in healthy controls, indicating that a second hit is required for such autoantibodies to induce or participate in pathology or alternatively that these antibodies are part of the natural antibody repertoire. In the present review, we describe autoantibodies against complement components and their functional consequences and discuss about their clinical relevance.

To understand the dynamic viral evolution observed during failure on raltegravir-containing regimens, we studied the genotypic and phenotypic patterns of resistance to raltegravir and the residual replication capacity (rRC) of HIV-1 variants selected in vivo.Clonal genotypic analyses were performed on sequential HIV-1 integrase sequences amplified from 11 failing patients and sampled every 4-24 weeks for up to 64 weeks. Fully replicating recombinant viruses were generated using modified vectors in which selected viral integrase genes amplified from patients' plasma were cloned. rRC was measured by a novel multiple cycle competition assay. Resistance to raltegravir and the rRC of resistant HIV-1 variants selected in vivo were evaluated in purified CD4+ T cells.In all of the patients but one, failure was associated with selection of mutations in positions 143, 148 or 155. Unlike mutations at position 143 (Y143S/K/R), identified alone or in combination with others, mutations at position 148 and 155 were always found in combination. A wide range of resistance levels to raltegravir [from 10- to 770-fold change in 50% inhibitory concentration (IC(50)) compared with baseline] was observed using recombinant viral clones. Finally, rRC was not significantly altered in highly resistant variants.Two patterns of viral evolution were observed in the resistant viral populations, driving the variants towards a fast (most of them with G140S + Q148H mutations) or progressive increase in resistance to raltegravir. These results may have implications either for the evaluation of genotypic results, or for the correct clinical use of the compound.

International audience; C3 glomerulopathy is a recently described form of CKD. C3GN is a subtype of C3 glomerulopathy characterized by predominant C3 deposits in the glomeruli and is commonly the result of acquired or genetic abnormalities in the alternative pathway (AP) of the complement system. We identified and characterized the first mutation of the C3 gene (p. I734T) in two related individuals diagnosed with C3GN. Immunofluorescence and electron microscopy studies showed C3 deposits in the subendothelial space, associated with unusual deposits located near the complement receptor 1 (CR1)-expressing podocytes. In vitro, this C3 mutation exhibited decreased binding to CR1, resulting in less CR1-dependent cleavage of C3b by factor 1. Both patients had normal plasma C3 levels, and the mutant C3 interacted with factor B comparably to wild-type (WT) C3 to form a C3 convertase. Binding of mutant C3 to factor H was normal, but mutant C3 was less efficiently cleaved by factor I in the presence of factor H, leading to enhanced C3 fragment deposition on glomerular cells. In conclusion, our results reveal that a CR1 functional deficiency is a mechanism of intraglomerular AP dysregu-lation and could influence the localization of the glomerular C3 deposits.

Introduction La frequence elevee des gammapathies monoclonales au cours de la glomerulopathie a depots de C3 (GPC3) chez les sujets de plus de 50 ans souleve la question de l’implication de l’Ig monoclonale dans l’activation de la voie alterne (VA). Materiels et methodes L’objectif de ce travail etait d’etudier les mecanismes d’activation de la VA chez 41 patients ayant une GPC3 et une Ig monoclonale. La recherche d’anticorps (Ac) diriges contre les proteines du complement a ete realisee par Elisa. L’activation de la C3 convertase en presence d’Ig totales (purification par proteine G) et/ou des fractions monoclonales et polyclonales (purification par chromatographie) a ete etudiee par WB et Elisa. Resultats Trente pour cent des patients ont un C3 diminue et 79 %, une augmentation du sC5b-9 temoins d’une activation de la C3/C5 convertase. La recherche d’anticorps diriges contre les proteines du complement (anti-CR1, anti-FH, anti-FI, anti-properdine et anti-C5) est positive chez 50 % des patients. Seuls 4 % des patients ont un C3Nef. Les Ac anti-FH et anti-CR1 sont associes a une diminution de l’activite cofacteur du FH et de CR1 chez 40 % des patients. Ces Ac sont polyclonaux et ne sont pas de la meme sous-classe que l’Ig monoclonale chez 75 % des patients testes. En presence de C3, FB et FD, les Ig totales jouent le role de « plateforme » et sont associees a un clivage plus important du C3 en C3b temoignant d’une augmentation de la formation de la C3 convertase, en phase fluide (12/32 patients) et/ou lorsqu’elles sont immobilisees (12/34 patients). Apres purification des Ig, la formation de la C3 convertase est plus importante en presence de la fraction monoclonale qu’en presence des Ig polyclonales chez 50 % des patients testes. En presence de serum humain normal (NHS), les Ig totales des patients sont associees a la formation accrue de C3a dans 30 % des cas. Un effet pro C5 convertase est observe en presence de properdine en exces chez les patients ayant des Ac anti-proteines regulatrices. Discussion Au cours de GPC3 associees aux Ig monoclonales, l’activation de la VA n’est pas liee, le plus souvent, a une activite Ac de l’Ig monoclonale. Conclusion L’activation non regulee de la VA resulte a la fois de mecanismes impliquant le composant monoclonal mais aussi les Ig polyclonales.

Dense deposit disease is characterized by dense deposits in the glomerular and tubular basement membranes. We report 3 cases with long-term follow-up differing in histologic pattern and clinical evolution. Clinical and histologic data were collected between 1976 and 2012. Age at the first manifestations was 6, 11, and 23 years, respectively. They included proteinuria (patient 1) and nephrotic syndrome (patients 2 and 3); renal function was normal in all cases. Two patients (1 and 3) had low complement component 3 (C3) levels. All patients had C3 nephritic factor. Genetic analysis revealed a rare variant of the factor I gene (patient 1) and a heterozygous mutation in complement factor H-related 5 gene (patient 2). Patient 1 underwent 3 biopsies during her 38 years of follow-up. Thickening of the capillary walls of the glomerular and tubular basement membranes was observed, with mild mesangial proliferation and progressive C3 and complement membrane attack complex mesangial deposits. However, renal function remained normal. Patient 2 also underwent 3 biopsies (22 years of follow-up), revealing a gradual decrease in C3 deposition and mesangial cell proliferation. He presented mild renal insufficiency. Patient 3 underwent 2 biopsies, which displayed unusual bulky membranous deposits, confirmed by electron microscopy, with no mesangial cell proliferation and little C3 and complement membrane attack complex deposits. Kidney function remained normal. These 3 cases of dense deposit disease differed in histologic pattern evolution: accumulation of C3 deposits, decrease in C3 deposits and proliferation, and isolated dense deposits. The histologic factors involved in clinical progression remain to be identified.

Atypical hemolytic uremic syndrome (aHUS) is a genetic ultrarare renal disease associated with overactivation of the alternative pathway of complement. Four gain-of-function mutations that form a hyperactive or deregulated C3 convertase have been identified in Factor B (FB) ligand binding sites. Here, we studied the functional consequences of 10 FB genetic changes recently identified from different aHUS cohorts. Using several tests for alternative C3 and C5 convertase formation and regulation, we identified two gain-of-function and potentially disease-relevant mutations that formed either an overactive convertase (M433I) or a convertase resistant to decay by FH (K298Q). One mutation (R178Q) produced a partially cleaved protein with no ligand binding or functional activity. Seven genetic changes led to near-normal or only slightly reduced ligand binding and functional activity compared with the most common polymorphism at position 7, R7. Notably, none of the algorithms used to predict the disease relevance of FB mutations agreed completely with the experimental data, suggesting that in silico approaches should be undertaken with caution. These data, combined with previously published results, suggest that 9 of 15 FB genetic changes identified in patients with aHUS are unrelated to disease pathogenesis. This study highlights that functional assessment of identified nucleotide changes in FB is mandatory to confirm disease association.

Introduction Le spectre des anomalies du complement associees aux glomerulopathies a depots de C3 (C3G) (maladie des depots denses et glomerulonephrite a depots predominants de C3) s’est considerablement elargi ces dernieres annees. L’objectif de cette etude est d’evaluer les caracteristiques immunologiques des patients de la cohorte francaise de C3G et leur implication en pratique clinique. Patients et methodes Deux cent quarante-sept patients ayant une C3G confirmee par la biopsie renale ont ete inclus avec : – exploration des proteines de la voie alterne (dosage de C3, C4, CH50, sC5b9, FH, FI, FB, recherche d’un C3NeF, d’un Ac anti-CFH, anti-C3, anti-CFB ou anti-properdine, recherche d’une anomalie des genes des CFH, CFI, MCP, C3, CFHR5 ; – recherche systematique d’une gammapathie monoclonale (IgMo) chez les plus de 50 ans. Discussion et conclusion Au diagnostic, 49 % des patients ont un SN impur associe a une IR dans 64 % des cas (eDFG moy 60 mL/min). Chez les plus de 50 ans, une IgMo est identifiee dans 67 % des cas et est associee a un pronostic renal nettement plus pejoratif (mediane de survie renale a 24 mois versus 80 mois dans les formes sans IgMo). Sur le plan immunologique, 38 % des patients ont un C3 bas et 31 %, un sC5b9 augmente. Les anomalies sont majoritairement acquises (66 % des cas) en rapport avec la presence d’un C3Nef (62 %) et/ou un Ac anti-FH (13 %) et/ou anti-C3 (3 %), et/ou anti-FB (4 %), et/ou anti-properdine (1 %). Une mutation sur un des genes de regulation de la voie alterne a ete identifiee dans 15 % des cas. Conclusion Les resultats 2014 de la cohorte francaise mettent en avant le caractere tres majoritairement acquis des C3G et la necessite de rechercher de nouvelles cibles antigeniques comme le C3 et le CFB. La presence d’une IgMo doit motiver l’instauration d’un traitement hematologique. L’eculizumab pourrait etre discute chez 30 % des patients ayant un sC5b9 augmente, temoin d’un clivage du C5.

Polyomaviruses KI (KIPyV) and WU (WUPyV) were described recently in children with acute respiratory disease. The pathogenic potential of these human viruses has not been determined completely, but a correlation between immunosuppression and virus reactivation has been suggested. In the present study, the association between KI/WUPyV infection and immunosuppression was investigated using sequential nasopharyngeal aspirates from asymptomatic adult hematopoietic stem cell transplant recipients. In parallel, an investigation on the WU/KIPyV prevalence in children with acute respiratory disease was also carried out. Two of the 126 samples obtained from the 31 hematopoietic transplant recipients were positive for KIPyV (1 sample, 0.79%) and WUPyV (1 sample, 0.79%). Both samples were obtained 15 days after allogeneic transplantation and virus persistence was not observed in subsequent samples. In symptomatic children, 7 of the 486 nasopharyngeal aspirates were positive for WUPyV (1.4%) and 1 for KIPyV (0.2%). Single polyomavirus infection was detected in four patients, whereas the remaining patients were co‐infected with respiratory syncityal virus (three patients) or adenovirus (one patient). The results suggest that WU/KIPyVs have a limited circulation in Italy and a low pathogenic potential in young children. Brief and asymptomatic infection can occur in hematopoietic transplant recipients. J. Med. Virol. 82:153–156, 2010. © 2009 Wiley‐Liss, Inc.

Objective : Evaluate HIV-1 subtype B integrase gene evolution in patients failing raltegravir (RAL)-based savage regimens by clonal analysis of the replicating viral quasispecies. Design : Seven triple class failure HIV-1 (subtype B)-infected patients, followed at San Raffaele Hospital and enrolled in the RAL Expanded Access Program (MK0518-023), were evaluated. Patients were followed up for 24-48 weeks and due to the absence of other active drugs, RAL was maintained in their regimens even if resistance mutations were detected. Methods : Immunologic and virologic parameters were recorded every 4 weeks, and amplification and clonal analysis of viral populations were performed at baseline and every 4-12 weeks in all patients. Results : Resistance to RAL appeared initially associated with selection of single variants (Y143R, Q148R N155H) in the majority of patients; however, in three patients, complex patterns of viral mutations were observed. The clonal analysis of viral quasispecies allowed to describe the evolution of each viral population and the progressive accumulation of RAL resistance-associated mutations and polymorphisms associated with therapy failure. Conclusion : The complex patterns of resistance mutations observed, including novel variants evolved under continuous RAL pressure, suggesting that they are the result of the equilibrium between drug resistance and enzyme function. Despite the efficacy of this compound, our data discourage its use in a functional monotherapy and maintaining RAL even in presence of RAL resistance-associated mutations may lead to the progressive formation of viral reservoirs with multiple integrase inhibitor-resistant variants that may limit the future efficacy of other integrase inhibitors due to cross-resistance.

The characteristics of intra-host human immunodeficiency virus type 1 (HIV-1) env evolution were evaluated in untreated HIV-1-infected subjects with different patterns of disease progression, including 2 normal progressor [NP], and 5 Long term non-progressor [LTNP] patients. High-resolution phylogenetic analysis of the C2-C5 env gene sequences of the replicating HIV-1 was performed in sequential samples collected over a 3–5 year period; overall, 301 HIV-1 genomic RNA sequences were amplified from plasma samples, cloned, sequenced and analyzed. Firstly, the evolutionary rate was calculated separately in the 3 codon positions. In all LTNPs, the 3rd codon mutation rate was equal or even lower than that observed at the 1st and 2nd positions (p = 0.016), thus suggesting strong ongoing positive selection. A Bayesian approach and a maximum-likelihood (ML) method were used to estimate the rate of virus evolution within each subject and to detect positively selected sites respectively. A great number of N-linked glycosylation sites under positive selection were identified in both NP and LTNP subjects. Viral sequences from 4 of the 5 LTNPs showed extensive positive selective pressure on the CD4-binding site (CD4bs). In addition, localized pressure in the area of the IgG-b12 epitope, a broad neutralizing human monoclonal antibody targeting the CD4bs, was documented in one LTNP subject, using a graphic colour grade 3-dimensional visualization. Overall, the data shown here documenting high selective pressure on the HIV-1 CD4bs of a group of LTNP subjects offers important insights for planning novel strategies for the immune control of HIV-1 infection.

A prospective 2‐year analysis including 322 infant patients with acute respiratory disease (ARD) hospitalized in a pediatric department in northern Italy was carried out to evaluate the role as respiratory pathogens or co‐pathogens of recently identified viruses. The presence of respiratory syncitial virus (RSV), human Metapneumoviruses (hMPVs), human Bocaviruses (hBoVs), and human Coronaviruses (hCoVs) was assayed by molecular detection and clinical symptoms evaluated. Nasopharyngeal aspirates from 150 of the 322 infants (46.6%) tested positive for at least one pathogen. Ninety samples (28.0%) tested positive for RSV RNA (61.5% genotype A and 38.5% genotype B), 46 (14.3%) for hMPV RNA (71.7% subtype A and 28.3% subtype B), 28 (8.7%) for hCoV RNA (39.3% hCoV‐OC43, 35.7% hCoV‐NL63, 21.4% hCoV‐HKU1, and 3.6% hCoV‐229E), and 7 (2.2%) for hBoV DNA (of the 6 typed, 50% subtype 1 and 50% subtype 2); 21/150 samples revealed the presence of 2 or more viruses. Co‐infection rates were higher for hMPVs, hCoVs, and hBoV (38.3%, 46.4%, and 57.1%,) and lower for RSV (23.3%). RSV was associated with the presence of complications (P

Introduction La glomerulonephrite a depots de C3 (GNC3) est associee le plus souvent a des anomalies acquises des proteines de la voie alterne (VA) du complement, de type C3Nef. Beaucoup plus rarement, des mutations des proteines regulatrices de la VA, comme le facteur H (FH) sont identifiees. Nous rapportons pour la premiere fois, le cas d’une forme familiale de GNC3 associee a une mutation heterozygote de C3, I734T, ainsi que les consequences fonctionnelles de cette mutation. Materiels et methodes La capacite du serum des patients a induire un defaut de regulation de la C3 convertase a ete etudiee grâce a 2 modeles cellulaires d’activation du complement : les tests hemolytiques et les cellules endotheliales glomerulaires. La proteine C3 recombinante a ete produite par un systeme de production eucaryote et les interactions de celle-ci avec le FH, CD46 et CR1 ont ete etudiees par resonance plasmonique de surface. L’activite cofacteur du FH, CR1 et de CD46 pour le clivage de C3 par le FI a ete etudiee par Western Blot. Resultats Le principal deficit observe en relation avec la mutation de C3 I734T est une diminution de la liaison de C3 au CR1, a l’origine d’une diminution de l’activite cofacteur de CR1. La liaison du C3 mute au FH est normale mais associee a un deficit de l’activite cofacteur du FH. En revanche la liaison a CD46 et son activite cofacteur sont normales. Enfin, le C3 mute interagit normalement avec le FB et forme une C3 convertase fonctionnelle. Discussion Nos resultats suggerent qu’un defaut de regulation par CR1 constitue un nouveau mecanisme responsable d’une activation intraglomerulaire de la VA, a l’origine des lesions de GNC3. De facon interessante, les 2 patients presentaient en outre d’importants depots de C3 et des debris membranaires inhabituels sur le versant extramembraneux. Compte tenu de l’expression podocytaire de CR1, comme unique proteine regulatrice sur ces cellules, nous suggerons que CR1 pourrait jouer un role dans la localisation specifique intrarenale des depots de C3. Conclusion CR1 constitue une proteine d’interet dans l’etude des GNC3.

Sequential nasopharyngeal aspirates from patients without respiratory symptoms undergoing hematopoietic stem cell transplantation (HSCT) were tested for genomic RNA of human metapneumovirus (hMPV). Persistent hMPV infection was documented in most of them and confirmed by virus isolation. hMPV infection etiology was also evaluated during the same period in samples from pediatric patients with acute respiratory diseases (ARDs). Sequence analysis of hMPV in HSCT recipients documented infection by hMPV genotype A and strong interhost similarity; this pattern differs from that observed in pediatric patients with ARDs. The data indicate that HSCT recipients may frequently develop symptomless hMPV infection.