RESUMEN
Identification of the molecular culprits of allergic reactions leveraged molecular allergology applications in clinical laboratory medicine. Molecular allergology shifted the focus from complex, heterogeneous allergenic extracts, e.g. pollen, food, or insect venom, towards genetically and immunologically defined proteins available for in vitro diagnosis. Molecular allergology is a precision medicine approach for the diagnosis, stratification, therapeutic management, follow-up and prognostic evaluation of patients within a large range of allergic diseases. Exclusively available for in vitro diagnosis, molecular allergology is nonredundant with any of the current clinical tools for allergy investigation. As an example of a major application, discrimination of genuine sensitization from allergen cross-reactivity at the molecular level allows the proper targeting of the culprit allergen and thus dramatically improves patient management. This review aims at introducing clinical laboratory specialists to molecular allergology, from the biochemical and genetic bases, through immunological concepts, to daily use in the diagnosis and management of allergic diseases.
RESUMEN
The complement receptor 1 (CR1) gene was shown to be involved in Alzheimer's disease (AD). We previously showed that AD is associated with low density of the long CR1 isoform, CR1*2 (S). Here, we correlated phenotype data (CR1 density per erythrocyte (CR1/E), blood soluble CR1 (sCR1)) with genetic data (density/length polymorphisms) in AD patients and healthy controls. CR1/E was enumerated using flow cytometry, while sCR1 was quantified by ELISA. CR1 polymorphisms were assessed using restriction fragment length polymorphism (RFLP), pyrosequencing, and high-resolution melting PCR. In AD patients carrying the H allele (HindIII polymorphism) or the Q allele (Q981H polymorphism), CR1/E was significantly lower when compared with controls carrying the same alleles (p < 0.01), contrary to sCR1, which was significantly higher (p < 0.001). Using multivariate analysis, a reduction of 6.68 units in density was associated with an increase of 1% in methylation of CR1 (estimate -6.68; 95% confidence intervals (CIs) -12.37, -0.99; p = 0.02). Our data show that, in addition to inherited genetic factors, low density of CR1/E is also acquired. The involvement of CR1 in the pathogenesis of AD might be linked to insufficient clearance of amyloid deposits. These findings may open perspectives for new therapeutic strategies in AD.
Asunto(s)
Enfermedad de Alzheimer/genética , Eritrocitos/patología , Receptores de Complemento 3b/sangre , Receptores de Complemento 3b/genética , Anciano , Anciano de 80 o más Años , Alelos , Sitios de Unión/genética , Estudios de Cohortes , Eritrocitos/química , Femenino , Genotipo , Humanos , Masculino , Metilación , Análisis Multivariante , Placa Amiloide/patología , Polimorfismo de Longitud del Fragmento de Restricción , Isoformas de Proteínas/sangre , Isoformas de Proteínas/genética , Factores de RiesgoRESUMEN
The receptor for advanced glycation end products (RAGE) is a pattern recognition receptor that interacts with advanced glycation end products, but also with C3a, CpG DNA oligonucleotides, and alarmin molecules such as HMGB1 to initiate a proinflammatory reaction. Systemic lupus erythematosus is an autoimmune disorder associated with the accumulation of RAGE ligands. We generated mice invalidated for RAGE in the lupus-prone B6-MRL Fas lpr/j background to determine the role of RAGE in the pathogenesis of systemic lupus erythematosus. We compared the phenotype of these mice with that of their wild-type and B6-MRL Fas lpr/j littermates. Lymphoproliferative syndrome, production of anti-dsDNA Abs, lupus nephritis, and accumulation of CD3(+)B220(+)CD4(-)CD8(-) autoreactive T cells (in the peripheral blood and the spleen) were significantly increased in B6-MRL Fas lpr/j RAGE(-/-) mice compared with B6-MRL Fas lpr/j mice (respectively p < 0.005, p < 0.05, p < 0.001, and p < 0.001). A large proportion of autoreactive T cells from B6-MRL Fas lpr/j mice expressed RAGE at their surface. Time course studies of annexin V expression revealed that autoreactive T cells in the spleen of B6-MRL Fas lpr/j-RAGE(-/-) mice exhibited a delay in apoptosis and expressed significantly less activated caspase 3 (39.5 ± 4.3%) than T cells in B6-MRL Fas lpr/j mice (65.5 ± 5.2%) or wild-type mice (75.3 ± 2.64%) (p = 0.02). We conclude that the deletion of RAGE in B6-MRL Fas lpr/j mice promotes the accumulation of autoreactive CD3(+)B220(+)CD4(-)CD8(-) T cells, therefore exacerbating lymphoproliferative syndrome, autoimmunity, and organ injury. This suggests that RAGE rescues the apoptosis of T lymphocytes when the death receptor Fas/CD95 is dysfunctional.
Asunto(s)
Apoptosis/inmunología , Nefritis Lúpica/inmunología , Trastornos Linfoproliferativos/inmunología , Receptores Inmunológicos/inmunología , Linfocitos T/inmunología , Animales , Antígenos de Diferenciación/genética , Antígenos de Diferenciación/inmunología , Apoptosis/genética , Caspasa 3/genética , Caspasa 3/inmunología , Eliminación de Gen , Regulación Enzimológica de la Expresión Génica/genética , Regulación Enzimológica de la Expresión Génica/inmunología , Nefritis Lúpica/genética , Nefritis Lúpica/patología , Trastornos Linfoproliferativos/genética , Trastornos Linfoproliferativos/patología , Ratones , Ratones Endogámicos MRL lpr , Ratones Noqueados , Receptor para Productos Finales de Glicación Avanzada , Receptores Inmunológicos/genética , Bazo/inmunología , Bazo/patología , Síndrome , Linfocitos T/patologíaRESUMEN
IgA nephropathy (IgAN), the most common primary glomerulonephritis worldwide, frequently leads to end-stage renal disease and kidney transplantation. However, disease recurrence often occurs after transplantation. Here we evaluated the predictive value of three markers for IgAN recurrence: the presence of galactose-deficient IgA1, IgG anti-IgA autoantibodies, and IgA-soluble (s) CD89 complexes. This was analyzed in 38 kidney transplant recipients with IgAN recurrence and compared with 22 patients transplanted for IgAN but without recurrence and with 17 healthy controls. Pre-transplantation galactose-deficient IgA1 serum levels were significantly higher in the recurrence compared with the no recurrence or control groups. IgA-IgG complexes were significantly elevated in the recurrence group. Both the recurrence and no recurrence groups had increased values of IgA-sCD89 complexes compared with healthy controls, but values were significantly lower in patients with recurrence compared with no recurrence. Areas under the receiver operating curve of the markers in pre-transplantation sera were 0.86 for galactose-deficient-IgA, 0.82 for IgA-IgG, and 0.78 for sCD89-IgA; all significant. Disease recurrence was associated with decreased serum galactose-deficient IgA1 and appearance of mesangial-galactose-deficient IgA1 deposits, whereas increased serum IgA-sCD89 complexes were associated with mesangial sCD89 deposits. Thus, galactose-deficient-IgA1, IgG autoantibodies, and IgA-sCD89 complexes are valuable biomarkers to predict disease recurrence, highlighting major pathogenic mechanisms in IgAN.
Asunto(s)
Complejo Antígeno-Anticuerpo/sangre , Antígenos CD/sangre , Autoanticuerpos/sangre , Glomerulonefritis por IGA/sangre , Inmunoglobulina A/sangre , Fallo Renal Crónico/sangre , Receptores Fc/sangre , Adulto , Anciano , Área Bajo la Curva , Biomarcadores/sangre , Biopsia , Estudios de Casos y Controles , Progresión de la Enfermedad , Femenino , Glomerulonefritis por IGA/diagnóstico , Glomerulonefritis por IGA/inmunología , Glomerulonefritis por IGA/cirugía , Humanos , Riñón/inmunología , Riñón/patología , Fallo Renal Crónico/diagnóstico , Fallo Renal Crónico/inmunología , Fallo Renal Crónico/cirugía , Trasplante de Riñón , Masculino , Persona de Mediana Edad , Valor Predictivo de las Pruebas , Curva ROC , Recurrencia , Factores de Riesgo , Resultado del TratamientoRESUMEN
Compact single-domain antibodies (sdAbs) are nearly 13 times smaller than full-size monoclonal antibodies (mAbs) and have a number of advantages for biotechnological applications, such as small size, high specificity, solubility, stability, and great refolding capacity. Carcinoembryonic antigen (CEA) is a tumor-associated glycoprotein expressed in a variety of cancers. Detection of CEA on the tumor cell surface may be carried out using anti-CEA antibodies and conventional fluorescent dyes. Semiconductor quantum dots (QDs) are brighter and more photostable than organic dyes; they provide the possibility for labeling of different recognition molecules with QDs of different colors but excitable with the same wavelength of excitation. In this study, the abilities for specific detection of CEA expressed by tumor cells with anti-CEA sdAbs biotinylated in vitro and in vivo, as well as with anti-CEA mAbs biotinylated in vitro, were compared using flow cytometry and the conjugates of streptavidin with QDs (SA-QDs). The results demonstrated that either in vitro or in vivo biotinylated anti-CEA sdAbs are more sensitive for cell staining compared to biotinylated anti-CEA mAbs. The data also show that simultaneous use of biotinylated sdAbs with highly fluorescent SA-QDs can considerably improve the sensitivity of detection of CEA on tumor cell surfaces.
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Antígeno Carcinoembrionario/análisis , Puntos Cuánticos/química , Anticuerpos de Dominio Único/química , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/química , Biotinilación , Línea Celular , Citometría de Flujo/métodos , Humanos , Ratones , Datos de Secuencia MolecularRESUMEN
HLA-B*15:648 differs from HLA-B*15:02:01:01 by one nucleotide substitution in codon 77 in exon 2.
Asunto(s)
Genes MHC Clase I , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Alelos , Exones/genética , Antígenos HLA-B/genéticaRESUMEN
HLA-DQA1*04:14N differs from HLA-DQA1*04:01:01:03 by a single nucleotide deletion in codon 113 in exon 3.
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Alelos , Exones , Cadenas alfa de HLA-DQ , Secuenciación de Nucleótidos de Alto Rendimiento , Prueba de Histocompatibilidad , Humanos , Cadenas alfa de HLA-DQ/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Prueba de Histocompatibilidad/métodos , Codón , Secuencia de Bases , Análisis de Secuencia de ADN/métodos , Eliminación de SecuenciaAsunto(s)
Alérgenos/inmunología , Hipersensibilidad/diagnóstico , Inmunoglobulina E/sangre , Venenos de Avispas/inmunología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Animales , Niño , Preescolar , Femenino , Francia , Humanos , Hipersensibilidad/sangre , Hipersensibilidad/inmunología , Inmunoglobulina E/inmunología , Lactante , Masculino , Persona de Mediana Edad , Avispas/inmunología , Adulto JovenRESUMEN
HLA-A*01:383 differs from HLA-A*01:01:01:01 by two nucleotide substitutions at positions 28 and 48 in exon 1.
Asunto(s)
Secuenciación de Nucleótidos de Alto Rendimiento , Nucleótidos , Humanos , Alelos , Exones/genética , Antígenos HLA-A/genéticaRESUMEN
HLA-A*33:220 differs from HLA-A*33:03:01:01 by one nucleotide substitution in codon 245 in exon 4.
RESUMEN
BACKGROUND: Rituximab (RTX) has been shown to be effective and safe for short-term treatment of severe pemphigus. Its long-term results remain unknown. OBJECTIVE: We sought to evaluate long-term RTX efficacy and safety in comparison with classic immunosuppressants for the treatment of severe pemphigus. METHODS: This retrospective study included, from 1997 to 2010, 24 consecutive patients with severe pemphigus, treated with RTX (n = 13) or systemic corticosteroids alone or combined with immunosuppressants (n = 11 control subjects). Anti-desmoglein antibodies were titered by enzyme-linked immunosorbent assay, every 3 months the first year, then at least annually. RESULTS: Among the 13 patients treated with RTX, 9 achieved complete remission 3 months after a first RTX cycle. Thereafter, 7 patients (4 with maintenance therapy) relapsed within a mean of 18 months after the last RTX cycle and received 1 or 2 additional RTX cycles. With mean follow-up at 41 months after the first RTX cycle and 28 months after the last one, all 13 patients remained in complete remission (5 patients off therapy). No severe RTX side effects occurred. Anti-desmoglein-3 autoantibodies remained positive in 7 patients, despite long-term complete remission. Long-term remission rates and immunologic profiles did not differ between patients with pemphigus according to RTX status. LIMITATIONS: This was a single-center, retrospective study. CONCLUSIONS: RTX appeared to be an effective and well-tolerated treatment for severe pemphigus at long term. However, the long-term remission rate without maintenance therapy did not differ significantly from that of control subjects. Anti-desmoglein-1 autoantibody titers were more reliable than anti-desmoglein-3 titers for long-term follow-up.
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Anticuerpos Monoclonales de Origen Murino/administración & dosificación , Factores Inmunológicos/administración & dosificación , Pénfigo/tratamiento farmacológico , Pénfigo/inmunología , Adolescente , Adulto , Anciano , Autoanticuerpos/sangre , Niño , Preescolar , Desmogleína 3/inmunología , Quimioterapia Combinada/métodos , Femenino , Estudios de Seguimiento , Humanos , Inmunosupresores/administración & dosificación , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , Rituximab , Índice de Severidad de la Enfermedad , Factores de Tiempo , Resultado del Tratamiento , Adulto JovenRESUMEN
Common strategy for diagnostics with quantum dots (QDs) utilizes the specificity of monoclonal antibodies (mAbs) for targeting. However QD-mAbs conjugates are not always well-suited for this purpose because of their large size. Here, we engineered ultrasmall nanoprobes through oriented conjugation of QDs with 13-kDa single-domain antibodies (sdAbs) derived from llama IgG. Monomeric sdAbs are 12 times smaller than mAbs and demonstrate excellent capacity for refolding. sdAbs were tagged with QDs through an additional cysteine residue integrated within the C terminal of the sdAb. This approach allowed us to develop sdAbs-QD nanoprobes comprising four copies of sdAbs coupled with a QD in a highly oriented manner. sdAbs-QD conjugates specific to carcinoembryonic antigen (CEA) demonstrated excellent specificity of flow cytometry quantitative discrimination of CEA-positive and CEA-negative tumor cells. Moreover, the immunohistochemical labeling of biopsy samples was found to be comparable or even superior to the quality obtained with gold standard protocols of anatomopathology practice. sdAbs-QD-oriented conjugates as developed represent a new generation of ultrasmall diagnostic probes for applications in high-throughput diagnostic platforms. FROM THE CLINICAL EDITOR: The authors report the development of sdAbs-QD-oriented conjugates, comprised of single domain antibodies that are 12 times smaller than regular mAb-s and quantum dots. These ultrasmall diagnostic probes represent a new generation of functionalized ODs for applications in high-throughput diagnostic platforms.
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Inmunoglobulina G/química , Sondas Moleculares/química , Puntos Cuánticos , Anticuerpos de Cadena Única/química , Animales , Camélidos del Nuevo Mundo , Antígeno Carcinoembrionario/química , Línea Celular Tumoral , Humanos , Neoplasias/diagnósticoRESUMEN
HLA-C*16:184 differs from HLA-C*16:02:01:01 by one nucleotide substitution at position 737 in exon 3.
Asunto(s)
Genes MHC Clase I , Antígenos HLA-C , Alelos , Exones/genética , Antígenos HLA-C/genética , Secuenciación de Nucleótidos de Alto Rendimiento , HumanosRESUMEN
HLA-B*53:64 differs from HLA-B*53:01:01:01 by one nucleotide substitution at position 1617 in exon 4.
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Antígenos HLA-B , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Alelos , Antígenos HLA-B/genética , Exones/genética , Genes MHC Clase IRESUMEN
HLA-C*03:04:94 differs from HLA-C*03:04:01:01 by one nucleotide substitution at position 737 in exon 4.
Asunto(s)
Antígenos HLA-C , Secuenciación de Nucleótidos de Alto Rendimiento , Alelos , Exones/genética , Genes MHC Clase I , Antígenos HLA-C/genética , HumanosRESUMEN
HLA-C*07:1001N differs from HLA-C*07:01:01:01 by a deletion of 17 nucleotides in exon 1.
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Antígenos HLA-C , Secuenciación de Nucleótidos de Alto Rendimiento , Alelos , Secuencia de Bases , Antígenos HLA-C/genética , Prueba de Histocompatibilidad , HumanosRESUMEN
HLA-C*05:255 differs from HLA-C*05:01:01:02 by one nucleotide substitution at position 2013 in exon 5.
Asunto(s)
Genes MHC Clase I , Antígenos HLA-C , Alelos , Exones/genética , Antígenos HLA-C/genética , Secuenciación de Nucleótidos de Alto Rendimiento , HumanosRESUMEN
Abnormal elevation of thyroid antibodies in the CSF is observed in 62-75% of Hashimoto's encephalopathy cases. However, the relationship between CSF thyroid antibody levels and response to therapy has been poorly evaluated. We report the case of a 68-year-old man with Hashimoto's encephalopathy, in whom there was a relation between the favorable clinical outcome and the disappearance of antithyroid antibodies from the CSF and a decrease in serum thyroid antibodies.
Une élévation anormale des anticorps thyroïdiens dans le LCR est observée dans 62 à 75 % des cas d'encéphalopathie de Hashimoto. Cependant, la relation entre les niveaux d'anticorps thyroïdiens dans le LCR et la réponse au traitement a été rarement évaluée. Nous rapportons le cas d'un homme de 68 ans atteint d'encéphalopathie de Hashimoto, chez qui l'évolution clinique favorable sous traitement était associée à la disparition des anticorps antithyroïdiens du LCR et une diminution des anticorps thyroïdiens sériques.
Asunto(s)
Encefalitis , Enfermedad de Hashimoto , Masculino , Humanos , Anciano , Enfermedad de Hashimoto/diagnóstico , Encefalitis/diagnóstico , AutoanticuerposRESUMEN
Semiconductor quantum dots (QDs) are proved to be unique fluorescent labels providing excellent possibilities for high-throughput detection and diagnostics. To explore in full QDs' advantages in brightness, photostability, large Stokes shift, and tunability by size fluorescence emission, they should be rendered stable in biological fluids and tagged with the target-specific capture molecules. Ideal QD-based nanoprobes should not exceed 15nm in diameter and should contain on their surface multiple copies of homogeneously oriented highly active affinity molecules, for example, antibodies (Abs). Direct conjugation of QDs with the Abs through cross-linking of QDs' amines with the sulfhydryl groups issued from the reduced Abs' disulfide bonds is the common technique. However, this procedure often generates conjugates in which the number of functionally active Abs on the surface of QDs does not always conform to expectations and is often low. Here we have developed an advanced procedure with the optimized critical steps of Ab reduction, affinity purification, and QD-Ab conjugation. We succeeded in reducing the Abs in such a way that the reduction reaction yields highly functional, partially cleaved, 75-kDa heavy-light Ab fragments. Affinity purification of these Ab fragments followed by their tagging with the QDs generates QD-Ab conjugates with largely improved functionality compared with those produced according to the standard procedures. The developed approach can be extended to conjugation of any type of Ab with different semiconductor, noble metal, or magnetic nanocrystals.