RESUMO
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.
Assuntos
Doença Antimembrana Basal Glomerular/diagnóstico , Autoantígenos/imunologia , Colágeno Tipo IV/imunologia , Técnica Indireta de Fluorescência para Anticorpo/métodos , Epitopos Imunodominantes/imunologia , Rim/patologia , Pulmão/patologia , Idoso , Doença Antimembrana Basal Glomerular/imunologia , Autoanticorpos/sangue , Autoantígenos/isolamento & purificação , Colágeno Tipo IV/isolamento & purificação , Testes Diagnósticos de Rotina , Feminino , Humanos , Imunoensaio , Epitopos Imunodominantes/isolamento & purificação , Masculino , Conformação ProteicaRESUMO
Suspicion of myelodysplastic syndromes (MDS) is one of the commonest reasons for bone marrow aspirate in elderly patients presenting with persistent peripheral blood (PB) cytopenia of unclear etiology. A PB assay that accurately rules out MDS would have major benefits. The diagnostic accuracy of the intra-individual robust coefficient of variation (RCV) for neutrophil myeloperoxidase (MPO) expression measured by flow cytometric analysis in PB was evaluated in a retrospective derivation study (44 MDS cases and 44 controls) and a prospective validation study (68 consecutive patients with suspected MDS). Compared with controls, MDS cases had higher median RCV values for neutrophil MPO expression (40.2% vs 30.9%; P<0.001). The area under the receiver operating characteristic curve estimates were 0.94 [95% confidence interval (CI): 0.86-0.97] and 0.87 (95%CI: 0.76-0.94) in the derivation and validation studies, respectively. A RCV lower than 30% ruled out MDS with 100% sensitivity (95%CI: 78-100%) and 100% negative predictive value (95%CI: 83-100%) in the prospective validation study. Neutrophil MPO expression measured by flow cytometric analysis in PB might obviate the need for invasive bone marrow aspirate and biopsy for up to 29% of patients with suspected MDS.
Assuntos
Biomarcadores Tumorais/análise , Citometria de Fluxo/métodos , Leucemia Mielomonocítica Crônica/diagnóstico , Síndromes Mielodisplásicas/diagnóstico , Neutrófilos/enzimologia , Peroxidase/metabolismo , Idoso , Estudos de Casos e Controles , Diagnóstico Diferencial , Feminino , Seguimentos , Humanos , Leucemia Mielomonocítica Crônica/enzimologia , Masculino , Síndromes Mielodisplásicas/enzimologia , Prognóstico , Estudos Prospectivos , Curva ROC , Estudos RetrospectivosRESUMO
The complement system has been implicated in several kidney diseases, such as antibody-mediated rejection after kidney transplantation. Antibody-depletion techniques allow successful ABO- and/or HLA-incompatible transplantation. Considering the IgG removal, the use of semi-selective immunoadsorption (IA) has been advocated. However, because of results on incomplete IgM depletion, the adjunctive use of membrane filtration (MF) has been proposed to enhance the removal of macromolecules and to interfere with complement activation. This secondary endpoint analysis of a recently published randomized, controlled, cross-over trial was designed to investigate the effect of combined treatment IA + MF compared to IA alone on complement depletion. Two treatment sequences, a single session of IA + MF followed by IA (and vice versa), were analyzed with regard to C5b-9, properdin, and mannose-binding lectin (MBL) levels. Neither IA alone nor IA + MF provoked complement activation as demonstrated by stable low levels of C5b-9 after the procedure as compared to the previous. The combined treatment substantially lowered properdin (77% vs. 26% reduction, P < 0.0001) as well as MBL concentrations (81% vs. 11% reduction, P < 0.0001). Recovery of properdin and MBL levels appears to be longer after IA alone compared to IA + MF. Depletion of properdin and MBL levels may have potential clinical implications in the setting of kidney transplantation.
Assuntos
Remoção de Componentes Sanguíneos/métodos , Imunoglobulina M/imunologia , Falência Renal Crônica/imunologia , Falência Renal Crônica/cirurgia , Transplante de Rim/métodos , Membranas Artificiais , Sistema ABO de Grupos Sanguíneos , Adsorção , Adulto , Incompatibilidade de Grupos Sanguíneos , Ativação do Complemento , Proteínas do Sistema Complemento/imunologia , Estudos Cross-Over , Feminino , Humanos , Masculino , Lectina de Ligação a Manose/química , Pessoa de Meia-Idade , Properdina/imunologiaRESUMO
Mutations within the central region of prion protein (PrP) have been shown to be associated with severe neurotoxic activity similar to that observed with Dpl, a PrP-like protein. To further investigate this neurotoxic effect, we generated lines of transgenic (Tg) mice expressing three different chimeric PrP-Dpl proteins. Chi1 (amino acids 1-57 of Dpl replaced by amino acids 1-125 of PrP) and Chi2 (amino acids 1-66 of Dpl replaced by amino acids 1-134 of PrP) abrogated the pathogenicity of Dpl indicating that the presence of a N-terminal domain of PrP (23-134) reduced the toxicity of Dpl, as reported. However, when the amino acids 1-24 of Dpl were replaced by amino acids 1-124 of PrP, Chi3 Tg mice, which express the chimeric protein at a very low level, start developing ataxia at the age of 5-7 weeks. This phenotype was not counteracted by a single copy of full-length-PrP(c) but rather by its overexpression, indicating the strong toxicity of the chimeric protein Chi3. Chi3 Tg mice exhibit severe cerebellar atrophy with a significant loss of granule cells. We concluded that aa25 to aa57 of Dpl, which are not present in Chi1 and Chi2 constructs, confer toxicity to the protein. We tested this possibility by using the 25-57 Dpl peptide in primary culture of mouse embryo cortical neurons and found a significant neurotoxic effect. This finding identifies a protein domain that plays a role in mediating Dpl-related toxicity.
Assuntos
Ataxia/genética , Ataxia/patologia , Cerebelo/patologia , Príons/genética , Animais , Ataxia/metabolismo , Western Blotting , Proteínas Ligadas por GPI/química , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Imuno-Histoquímica , Camundongos , Camundongos Transgênicos , Microscopia Eletrônica de Transmissão , Príons/química , Príons/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Quimeras de TransplanteRESUMO
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.
Assuntos
COVID-19 , Subpopulações de Linfócitos , Linfopenia , Linfócitos B , Linfócitos T CD4-Positivos , Linfócitos T CD8-Positivos , COVID-19/diagnóstico , COVID-19/imunologia , COVID-19/mortalidade , Humanos , Ativação Linfocitária , Linfopenia/virologia , SARS-CoV-2RESUMO
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 Tyr(271) in this respect.
Assuntos
Carboidratos/química , Lectinas/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Polissacarídeos/metabolismo , Cristalografia por Raios X , Fibrinogênio/química , Fibrinogênio/genética , Humanos , Lectinas/química , Mutagênese Sítio-Dirigida , Mutação de Sentido Incorreto , Ácido N-Acetilneuramínico/química , Polissacarídeos/química , Análise Serial de Proteínas , Ligação Proteica , Tirosina , FicolinasRESUMO
A growing number of studies have investigated the interaction between C1q and PrP, but the oligomeric form of PrP involved in this interaction remains to be determined. Aggregation of recombinant full-length murine PrP in the presence of 100 mm NaCl allowed us to isolate three different types of oligomers by size-exclusion chromatography. In contrast to PrP monomers and fibrils, these oligomers activate the classical complement pathway, the smallest species containing 8-15 PrP protomers being the most efficient. We used Thioflavine T fluorescence to monitor PrP aggregation and showed that, when added to the reaction, C1q has a cooperative effect on PrP aggregation and leads to the formation of C1q-PrP complexes. In these complexes, C1q interacts through its globular domains preferentially with the smallest oligomers, as shown by electron microscopy, and retains the ability to activate the classical complement pathway. Using two cell lines, we also provide evidence that C1q inhibits the cytotoxicity induced by the smallest PrP oligomers. The cooperative interaction between C1q and PrP could represent an early step in the disease, where it prevents elimination of the prion seed, leading to further aggregation.
Assuntos
Complemento C1q/metabolismo , Príons/química , Amiloide/química , Animais , Benzotiazóis , Cromatografia/métodos , Complemento C4/química , Proteínas do Sistema Complemento , Humanos , Imunidade Inata , Camundongos , Microscopia Eletrônica/métodos , Neurônios/metabolismo , Ligação Proteica , Tiazóis/químicaRESUMO
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 Lys(55) in the collagenous region of MBL in the interaction with the MASPs and calreticulin (CRT). To test the possible involvement of the homologous residues Lys(57) of L-ficolin and Lys(47) 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 Lys(57) of L-ficolin and Lys(47) 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.
Assuntos
Calreticulina/metabolismo , Colágeno/fisiologia , Lectinas/fisiologia , Lisina/fisiologia , Serina Proteases Associadas a Proteína de Ligação a Manose/metabolismo , Glicoproteínas de Membrana/metabolismo , Receptores de Complemento/metabolismo , Animais , Sítios de Ligação/genética , Sítios de Ligação/imunologia , Células CHO , Linhagem Celular , Colágeno/química , Cricetinae , Cricetulus , Variação Genética , Humanos , Lectinas/biossíntese , Lectinas/química , Lectinas/genética , Lisina/química , Lectina de Ligação a Manose/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , FicolinasRESUMO
Sex effect on the incubation period of variant Creutzfeldt-Jakob disease (vCJD) disease in human and ME-7 murine models was investigated. In the 167 vCJD cases reported in the United Kingdom as of January 2009, age at onset was significantly lower in female patients (by 2 years) than in male patients after stratification on birth cohort. In C57/Bl6N mice infected with ME-7 scrapie strain, incubation was shorter in female than in male mice. The incubation period increased in castrated male mice after intraperitoneal infection but not after intracerebral inoculation. In the absence of androgen receptors, the incubation period for prion disease increased after intraperitoneal inoculation. In ovariectomized or estrogen receptor alpha-defective female mice, no effect was observed on the incubation period of mouse prion disease. These results show that androgens influence the prion diseases incubation period after inoculation at a peripheral site.
Assuntos
Período de Incubação de Doenças Infecciosas , Doenças Priônicas/fisiopatologia , Adulto , Fatores Etários , Androgênios/fisiologia , Animais , Castração , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Priônicas , Príons/administração & dosagem , Fatores SexuaisRESUMO
Background: The 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. Methods: We 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. Results: Four 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. Conclusion: These 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.
Assuntos
COVID-19/imunologia , COVID-19/mortalidade , Lectina de Ligação a Manose da Via do Complemento , SARS-CoV-2 , Adulto , Idoso , Idoso de 80 Anos ou mais , Proteínas do Sistema Complemento/imunologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos RetrospectivosRESUMO
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.
Assuntos
Aspergillus/imunologia , Lectina de Ligação a Manose da Via do Complemento/fisiologia , Esporos Fúngicos/imunologia , HumanosRESUMO
The complement is a powerful cascade of the innate immunity and also acts as a bridge between innate and acquired immune defence. Complement activation can occur via three distinct pathways, the classical, alternative and lectin pathways, each resulting in the common terminal pathway. Complement activation results in the release of a range of biologically active molecules that significantly contribute to immune surveillance and tissue homeostasis. Several soluble and membrane-bound regulatory proteins restrict complement activation in order to prevent complement-mediated autologous damage, consumption and exacerbated inflammation. The crucial role of complement in the host homeostasis is illustrated by association of both complement deficiency and overactivation with severe and life-threatening diseases. Autoantibodies targeting complement components have been described to alter expression and/or function of target protein resulting in a dysregulation of the delicate equilibrium between activation and inhibition of complement. The spectrum of diseases associated with complement autoantibodies depends on which complement protein and activation pathway are targeted, ranging from autoimmune disorders to kidney and vascular diseases. Nevertheless, these autoantibodies have been identified as differential biomarkers for diagnosis or follow-up of disease only in a small number of clinical conditions. For some autoantibodies, a clear relationship with clinical manifestations has been identified, such as anti-C1q, anti-Factor H, anti-C1 Inhibitor antibodies and C3 nephritic factor. For other autoantibodies, the origin and the functional consequences still remain to be elucidated, questioning about the pathophysiological significance of these autoantibodies, such as anti-mannose binding lectin, anti-Factor I, anti-Factor B and anti-C3b antibodies. The detection of autoantibodies targeting complement components is performed in specialized laboratories; however, there is no consensus on detection methods and standardization of the assays is a real challenge. This review summarizes the current panorama of autoantibodies targeting complement recognition proteins of the classical and lectin pathways, associated proteases, convertases, regulators and terminal components, with an emphasis on autoantibodies clearly involved in clinical conditions.
Assuntos
Doenças Autoimunes/etiologia , Doenças Autoimunes/metabolismo , Proteínas do Sistema Complemento/imunologia , Proteínas do Sistema Complemento/metabolismo , Suscetibilidade a Doenças/imunologia , Imunidade Inata , Animais , Autoanticorpos/imunologia , Autoantígenos/imunologia , Doenças Autoimunes/patologia , Autoimunidade , Ativação do Complemento/imunologia , Complemento C1q/imunologia , Epitopos/metabolismo , HumanosRESUMO
The innate immune response provides a first line of defense against common microorganisms and, for more complex and/or recurring situations where pathogens must be eliminated, an adaptive immune response has emerged and evolved to provide better protection against subsequent infections. However, such dichotomy has to be reevaluated because innate B cells (e.g., B1 and marginal zone B cells) and the newly described innate lymphoid cells (iLC) have been found to exhibit innate-like properties, such as antigen internalization, regulatory B cell functions, and helper T cell activities. In addition, the production and function of natural antibodies (nAbs) by innate B cells and their capacity to activate the classical complement pathway constitute additional important mechanisms at the junction of innate and adaptive immunity as well as the recent integration of platelets into the innate immune spectrum. There is no doubt that these mechanisms present an advantage in immunity and homeostasis particularly during the first years of life, but arguments are arising to consider that these precursors may have detrimental effects in a variety of autoimmune/inflammatory diseases, allergies and cancers, as well as in response to immunotherapy. Accordingly, and as presented in this special issue of Clinical Reviews in Allergy and Immunology, a better comprehension of the key molecular and cellular actors implicated at the crossroads of the innate and adaptive immune response represents a new challenge in our understanding of the immunological and immunopathological responses.
Assuntos
Imunidade Adaptativa , Sistema Imunitário/fisiologia , Imunidade Inata , Imunidade Adaptativa/efeitos dos fármacos , Animais , Subpopulações de Linfócitos B/efeitos dos fármacos , Subpopulações de Linfócitos B/imunologia , Subpopulações de Linfócitos B/metabolismo , Biomarcadores , Plasticidade Celular/imunologia , Epigênese Genética/efeitos dos fármacos , Humanos , Sistema Imunitário/efeitos dos fármacos , Imunidade Humoral , Imunidade Inata/efeitos dos fármacos , Inibidores de Janus Quinases/farmacologia , Transdução de Sinais/efeitos dos fármacos , Subpopulações de Linfócitos T/efeitos dos fármacos , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Receptores Toll-Like/metabolismoRESUMO
Civil society at large and all caregivers, whether at home or within institutions, are involved in palliative care However, procedures may vary considerably, excluding a single approach. So as to best adapt their responses, the authors recorded everyone's expectations. Such a participatory methodology is, sine 1990, behind the establishment of local networks providing assistance, support and training to physicians non-specialized in palliative care (general practitioners, specialists, students or residents facing specific aspects of this medical management, as well as other health and social workers).
Assuntos
Serviços de Assistência Domiciliar/organização & administração , Cuidados Paliativos/organização & administração , HumanosRESUMO
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.
Assuntos
Angioedemas Hereditários/metabolismo , Bradicinina/metabolismo , Proteína Inibidora do Complemento C1/análise , Algoritmos , Angioedema/induzido quimicamente , Angioedema/metabolismo , Angioedemas Hereditários/classificação , Inibidores da Enzima Conversora de Angiotensina/efeitos adversos , Criança , Comorbidade , Proteína Inibidora do Complemento C1/genética , Diagnóstico Precoce , Fator XII/fisiologia , Feminino , Fibrinolisina/fisiologia , Doenças Hematológicas/epidemiologia , Angioedema Hereditário Tipos I e II/diagnóstico , Angioedema Hereditário Tipos I e II/metabolismo , Humanos , Calicreínas/fisiologia , Lúpus Eritematoso Sistêmico/epidemiologia , Gravidez , Complicações na Gravidez/sangue , Avaliação de SintomasRESUMO
Transmissible spongiform encephalopathies are fatal neurodegenerative disorders thought to be transmitted by self-perpetuating conformational conversion of a neuronal membrane glycoprotein (PrP(C), for "cellular prion protein") into an abnormal state (PrP(Sc), for "scrapie prion protein"). Doppel (Dpl) is a protein that shares significant biochemical and structural homology with PrP(C). In contrast to its homologue PrP(C), Dpl is unable to participate in prion disease progression or to achieve an abnormal PrP(Sc)-like state. We have constructed a chimeric mouse protein, composed of the N-terminal domain of PrP(C) (residues 23-125) and the C-terminal part of Dpl (residues 58-157). This chimeric protein displays PrP-like biochemical and structural features; when incubated in presence of NaCl, the alpha-helical monomer forms soluble beta-sheet-rich oligomers which acquire partial resistance to pepsin proteolysis in vitro, as do PrP oligomers. Moreover, the presence of aggregates akin to protofibrils is observed in soluble oligomeric species by electron microscopy.
Assuntos
Fragmentos de Peptídeos/química , Proteínas PrPC/química , Príons/química , Animais , Proteínas Ligadas por GPI , Camundongos , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Proteínas PrPC/genética , Proteínas PrPC/metabolismo , Príons/genética , Príons/metabolismo , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Cloreto de Sódio/química , SoluçõesRESUMO
Lupus nephritis (LN) is one of the most frequent and severe manifestations of systemic lupus erythematosus (SLE), considered as the major predictor of poor prognosis. An early diagnosis of LN is a real challenge in the management of SLE and has an important implication in guiding treatments. In clinical practice, conventional parameters still lack sensitivity and specificity for detecting ongoing disease activity in lupus kidneys and early relapse of nephritis. LN is characterized by glomerular kidney injury, essentially due to deposition of immune complexes involving autoantibodies against cellular components and circulating proteins. One of the possible mechanisms of induction of autoantibodies in SLE is a defect in apoptotic cells clearance and subsequent release of intracellular autoantigens. Autoantibodies against soluble protective molecules involved in the uptake of dying cells, including complement proteins and pentraxins, have been described. In this review, we present the main autoantibodies found in LN, with a focus on the antibodies against these protective molecules. We also discuss their pathogenic role and conclude with their potential interest as serological biomarkers in LN.
Assuntos
Biomarcadores/sangue , Glomérulos Renais/anormalidades , Rim/patologia , Lúpus Eritematoso Sistêmico/genética , Nefrite Lúpica/imunologia , Feminino , Humanos , Lúpus Eritematoso Sistêmico/patologia , MasculinoRESUMO
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.
Assuntos
Células da Medula Óssea/citologia , Citometria de Fluxo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
OBJECTIVE: Systemic lupus erythematosus (SLE) is a multisystem inflammatory disease characterized by the production of various autoantibodies. The aim of this study was to investigate the presence of anti-ficolin-2 antibodies in SLE patients and to evaluate the association between the levels of these autoantibodies, clinical manifestations, and disease activity. METHODS: This is a comparative study using a cohort of 165 SLE patients and 48 healthy subjects. SLE patients were further divided into 2 groups (low disease activity [SLE Disease Activity Index (SLEDAI) score ≤4, n = 88] and high disease activity [SLEDAI score >4, n = 77]). Clinical manifestations were defined according to the physician in charge. Active lupus nephritis (LN) was documented by kidney biopsy. Detection of anti-ficolin-2 antibodies was performed by enzyme-linked immunosorbent assay. RESULTS: Levels of anti-ficolin-2 autoantibodies were significantly higher in SLE patients as compared to healthy subjects and associated with SLEDAI score. They were found to be positive in 61 of 165 SLE patients (37%). The presence of anti-ficolin-2 antibodies was significantly related only to renal involvement, with a very high prevalence (86%) of anti-ficolin-2 antibodies in SLE patients with active LN. Patients with active proliferative LN had significantly more positive anti-ficolin-2 antibodies than those with nonproliferative LN. The combination of anti-ficolin-2, anti-ficolin-3, and anti-C1q demonstrated a very high specificity (98%) for the diagnosis of active LN. CONCLUSION: Our results support the usefulness of anti-ficolin-2 as a complementary serologic biomarker for the diagnosis of active lupus with renal manifestations.
Assuntos
Anticorpos Antinucleares/sangue , Autoanticorpos/sangue , Lectinas/metabolismo , Lúpus Eritematoso Sistêmico/sangue , Nefrite Lúpica/sangue , Adulto , Autoanticorpos/imunologia , Biomarcadores/metabolismo , Biópsia por Agulha , Ensaio de Imunoadsorção Enzimática/métodos , Feminino , Humanos , Imuno-Histoquímica , Lúpus Eritematoso Sistêmico/diagnóstico , Lúpus Eritematoso Sistêmico/epidemiologia , Nefrite Lúpica/diagnóstico , Nefrite Lúpica/epidemiologia , Masculino , Pessoa de Meia-Idade , Prevalência , Sensibilidade e Especificidade , Índice de Gravidade de Doença , FicolinasRESUMO
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.