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OBJECTIVES: Spondyloarthritis (SpA) is a chronic inflammatory disease of unknown aetiology. Previously, we identified autoantibodies against CD74 in sera of SpA patients. The aim of this study was to evaluate CD74 as a T cell antigen in SpA. METHODS: Recombinant CD74 protein and a panel of selected peptides representing its amino acid residues were examined for their capability to stimulate peripheral blood mononuclear cells from patients with SpA. In particular, cytokine production by CD4+ T cells was evaluated with flow cytometric detection of intracellular TNF-α, IFNγ, TGFß and IL-17A. Patients' sera were tested for antibodies against CD74 using ELISA. Samples from patients with rheumatoid arthritis and healthy blood donors were similarly tested as controls. RESULTS: Significantly more CD4+ T cells from SpA patients produced TNF-α, IFNγ and IL-17A in response to recombinant CD74 than patients with rheumatoid arthritis or healthy blood donors. Among evaluated epitopes, the most promiscuous one lies within the peptide of the amino acid residues 142-185, which appeared more immunogenic. Further, the proportion of cytokine producing CD4+ T cells was significantly higher among SpA patients with autoantibodies against CD74. CONCLUSIONS: CD74 is a T cell antigen in SpA, eliciting Th1 and Th17 responses, which may be relevant in disease pathogenesis. Recognition of the highly immunogenic amino acid residues of CD74 may contribute to our understanding of autoimmune responses of T helper cells in SpA.
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Antígenos de Diferenciación de Linfocitos B/inmunología , Autoanticuerpos/inmunología , Espondiloartritis , Artritis Reumatoide/diagnóstico , Artritis Reumatoide/inmunología , Biomarcadores , Estudios de Casos y Controles , Femenino , Humanos , Leucocitos Mononucleares , Masculino , Persona de Mediana Edad , Espondiloartritis/diagnóstico , Espondiloartritis/inmunología , Células Th17RESUMEN
Sarcoidosis is characterized by multiorgan involvement and granulomatous inflammation. Its origin is unknown and the potential role of autoimmunity has not been sufficiently determined. We investigated the presence of autoantibodies in sarcoidosis using protein array technology. The derivation cohort consisted of patients with sarcoidosis (n = 25) and controls including autoimmune disease and blood donors (n = 246). In addition, we tested a validation cohort including pulmonary sarcoidosis patients (n = 58) and healthy controls (n = 13). Initially, sera of three patients with sarcoidosis were screened using a protein array with 28.000 proteins against controls. Thereby we identified the Negative Elongation Factor E (NELF-E) as an autoantigen. With confirmatory Enzyme-linked Immunosorbent Assay (ELISA)testing, 29/82 patients (35%) with sarcoidosis had antibodies against NELF-E of the Immunoglobulin (Ig) G type, whereas 18/253 (7%) sera of the controls were positive for NELF-E. Clinically, there was an association of the frequency of NELF-E antibody detection with lung parenchymal involvement and corresponding x-ray types. NELF-E autoantibodies are associated with sarcoidosis and should be further investigated.
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OBJECTIVE: The aim of this study is to analyse the influence of LILRA3 and the genetic leukocyte immunoglobulin-like receptor 3 (LILRA3) deletion on transmission and clinical course of HIV infection. DESIGN: Case and control study. METHODS: LILRA3 genotypes were determined by PCR. HIV patients were categorized into short-term progressors, normal progressors and long-term nonprogressors according to the clinical course. Functional studies were performed using real-time PCR, intracellular flow cytometry and ELISA. RESULTS: The prevalence of the homozygous LILRA3 deletion was higher in HIV-positive individuals (nâ=â439) than in controls (nâ=â651) (Pâ=â0.02). The disease progression was faster in homozygously deleted patients with more short-term progressors than in heterozygous (Pâ=â0.03) and homozygously positive (Pâ=â0.002) individuals. These results have been confirmed in a seroconverter cohort (nâ=â288). The frequency of the homozygous deletion in the confirmation cohort was higher than in controls (Pâ=â0.04). Combining both cohorts, the proportion of homozygously LILRA3-deleted individuals was 6.2% in HIV-infected patients (nâ=â727) vs. 3.2% in controls (Pâ=â0.01). Functional analysis revealed an upregulation of the LILRA3 gene in real-time PCR in treated patients when compared with untreated patients (Pâ=â0.007) and controls (Pâ=â0.02) resulting in a higher LILRA3 expression in CD4 (Pâ=â0.008) and CD14 (Pâ=â0.02) cells of untreated patients in intracellular flow cytometry. LILRA 3 concentrations in the sera were similar between the groups, in untreated patients a correlation between viral load and LILRA3 concentration was found. CONCLUSION: The homozygous LILRA3 deletion is associated with a higher susceptibility for HIV disease and with a faster disease progression.
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Predisposición Genética a la Enfermedad , Infecciones por VIH/genética , Receptores Inmunológicos/deficiencia , Eliminación de Secuencia , Adulto , Estudios de Casos y Controles , Ensayo de Inmunoadsorción Enzimática , Femenino , Citometría de Flujo , Infecciones por VIH/patología , Infecciones por VIH/transmisión , Humanos , Masculino , Reacción en Cadena de la Polimerasa , Receptores Inmunológicos/genética , Factores de RiesgoRESUMEN
The innovative medicine initiative project called PRECISESADS will study 2.500 individuals affected by systemic autoimmune diseases (SADs) and controls. Among extensive OMICS approaches, multi-parameter flow cytometry analyses will be performed in eleven different centers. Therefore, the integration of all data in common bioinformatical and biostatistical investigations requires a fine mirroring of all instruments. We describe here the procedure elaborated to achieve this prerequisite. One flow cytometer chosen as reference instrument fixed the mean fluorescence intensities (MFIs) of 8 different fluorochrome-conjugated antibodies (Abs) using VersaComp Ab capture beads. The ten other centers adjusted their own PMT voltages to reach the same MFIs. Subsequently, all centers acquired Rainbow 8-peak beads data on a daily basis to follow the stability of their instrument overtime. One blood sample has been dispatched and concomitantly stained in all centers. Comparison of leukocytes frequencies and cell surface marker MFIs demonstrated the close sensitivity of all flow cytometers, allowing a multicenter analysis. The effective multi-center harmonization enables the constitution of a workable wide flow cytometry database for the identification of specific molecular signatures in individuals with SADs.
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Enfermedades Autoinmunes/clasificación , Citometría de Flujo/instrumentación , Anticuerpos , Enfermedades Autoinmunes/sangre , Calibración , Europa (Continente) , Citometría de Flujo/normas , HumanosRESUMEN
NK cell function is important in the immune response to HIV infection. NKG2C and NKG2A are activating and inhibitory NK cell receptors, respectively, and their only known ligand, HLA-E, demonstrates increased expression in HIV infection and presents at least one HIV-derived peptide. A variation in chromosome 12 exists in which the 16-kb section of DNA encompassing the nkg2c gene is completely absent. DNA samples of 433 HIV-1-infected patients and 280 controls were genotyped by PCR, and revealed an association of the absence variation with a higher risk of HIV infection, as well as faster progression and higher pretreatment viral loads (p<0.05, respectively). Surface NKG2C expression, analyzed by FACS, on the freshly isolated lymphocytes of 20 control and 19 HIV-infected donors revealed that NKG2C expression is genotype dependent in both populations: no NKG2C expression in the -/- groups, intermediate expression in the +/- groups, and highest expression in the +/+ groups. The comparison of NKG2C and NKG2A expression in HIV and control groups (+/- and +/+ included) indicates an increased NKG2C expression on HIV patient NK cells (p<0.05) and decreased inhibitory NKG2A expression on CD8 T cells (p<0.001), and both these effects are more striking in the +/+ genotype (p<0.005). Furthermore, a positive correlation was found between HIV viral load and the proportion of NKG2C(+) NK cells. The increased expression of NKG2C in HIV patients, in combination with the genetic association of the absence variation with an increased susceptibility to HIV infection, higher HIV viral set point, and a faster progression, indicate that NKG2C is important in the defense against HIV infection and progression.
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Infecciones por VIH/genética , VIH-1 , Células Asesinas Naturales/inmunología , Subfamília C de Receptores Similares a Lectina de Células NK/genética , Eliminación de Secuencia , Adolescente , Adulto , Anciano , Niño , Cromosomas Humanos Par 12 , Femenino , Expresión Génica , Infecciones por VIH/inmunología , Infecciones por VIH/metabolismo , Humanos , Células Asesinas Naturales/metabolismo , Masculino , Persona de Mediana Edad , Subfamília C de Receptores Similares a Lectina de Células NK/metabolismo , Factores de Riesgo , Análisis de Secuencia de ADN , Carga Viral , Adulto JovenRESUMEN
OBJECTIVE: In systemic lupus erythematosus (SLE), immune complexes (ICs) containing pathogenic IgG anti-double-stranded DNA (anti-dsDNA) autoantibodies are deposited in renal capillaries and initiate glomerulonephritis (GN) by the activation of complement and effector cells. In contrast, it has been demonstrated that the presence of IgM anti-dsDNA antibodies correlates negatively with the development of GN in SLE. The aim of this study was to determine whether anti-dsDNA antibodies of the IgM isotype protect against IC-mediated organ damage in SLE. METHODS: Lupus-prone (NZB x NZW)F(1) mice (females) were treated with murine monoclonal IgM anti-dsDNA antibodies. Treatment was delivered by subcutaneous injection at a dosage of 100 mug/week starting at 16 weeks of age (prophylactic) or at 24 weeks of age (therapeutic). RESULTS: Mice treated with IgM anti-dsDNA exhibited a delayed onset of proteinuria and a reduced degree of renal pathology, which resulted in significantly improved survival as compared with control mice. Serum concentrations of IgG anti-dsDNA antibodies were not significantly modified. However, glomerular deposition of ICs was markedly reduced in both treatment protocol groups. In contrast, higher amounts of IgG and IgM and increased expression of Fcgamma receptor were demonstrated in liver sections from the treated mice compared with the untreated mice, suggesting an enhanced clearance of soluble ICs from phagocytic cells of the reticuloendothelial system. CONCLUSION: These data demonstrate the efficacy of IgM anti-dsDNA treatment in inhibiting the pathologic changes of lupus in (NZB x NZW)F(1) mice. Lower glomerular IC deposition is associated with a reduced inflammatory response and impaired organ damage. The reduced frequency of GN in SLE patients who have IgM anti-dsDNA antibodies may therefore reflect a disease-modifying effect of this class of autoantibodies that has potential therapeutic implications. Our findings should encourage the development of new therapeutic modalities using IgM anti-dsDNA antibodies in humans with SLE.