RESUMEN
Recombinant Abs are gaining increasing importance for the treatment of certain cancers or immunological or neurologic disorders. The ELISA is one of the most used analytical tools for detecting and quantifying Abs of interest. However, the performance of ELISAs often varies because of nonstandard experimental procedures as well as inadequate data analysis. In our study, we standardized a procedure and statistical analysis for a highly sensitive ELISA of a mouse Ab in mouse (C57BL/6J) CNS tissue. The following steps are of crucial importance: 1) calculation of the limit of detection based on control tissue lysate samples in the same testing buffer as the testing samples; 2) calculation of the limit of quantification as measured with acceptable accuracy and precision; and 3) a five-parameter logistic regression model to interpolate the symmetric and asymmetric standard curves. We also show that three amplification Abs can significantly increase the sensitivity of the ELISA compared with a two amplification Ab setup. This standardized procedure may be a valuable tool to increase the sensitivity, reproducibility, and precision of ELISA studies in basic science and translational research.
Asunto(s)
Anticuerpos , Sistema Nervioso Central , Animales , Ratones , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Ratones Endogámicos C57BL , Ensayo de Inmunoadsorción Enzimática/métodosRESUMEN
BACKGROUND: The Rotarod test with commercial apparatus is widely used to assess locomotor performance, balance and motor learning as well as the deficits resulting from diverse neurological disorders in laboratory rodents due to its simplicity and objectivity. Traditionally, the test ends when rodents drop from the accelerating, turning rod, and the only parameter used commonly is "latency to fall". The values of individual animals can often vary greatly. RESULTS: In the present study, we established a procedure for mice with 4 consecutive days of training with 4 trials per day and modified the testing procedure by placing the mice back on the rod repeatedly after each fall until the trial ends (5 min). Data from the fourth training day as baseline results showed that the second, third and fourth trial were more consistent than the first, probably due to habituation or learning. There was no difference between the second, third and fourth trial, two trials may be sufficient in testing. We also introduced 3 additional read-outs: Longest duration on the rod (s), Maximal distance covered (cm), and Number of falls to better evaluate the motor capacity over the 5 min of testing. We then used this 4-parameter analysis to capture the motor deficits of mice with mild to moderate traumatic brain injuries (by a weight dropping on the skull (Marmarou model)). We found that normalization of data to individual baseline performance was needed to reduce individual differences, and 4 trials were more sensitive than two to show motor deficits. The parameter of Maximal distance was the best in detecting statistically significant long-term motor deficits. CONCLUSIONS: These results show that by making adjustments to the protocol and employing a more refined analysis, it is possible to expand a widely used routine behavioral test with additional accessible parameters that detect relevant deficits in a model of mild to moderate traumatic brain injury. The modified Rotarod test maybe a valuable tool for better preclinical evaluations of drugs and therapies.
Asunto(s)
Cabeza , Aprendizaje , Animales , Ratones , Prueba de Desempeño de Rotación con Aceleración Constante , CráneoRESUMEN
Stroke is a major cause of serious disability due to the brain's limited capacity to regenerate damaged tissue and neuronal circuits. After ischemic injury, a multiphasic degenerative and inflammatory response is coupled with severely restricted vascular and neuronal repair, resulting in permanent functional deficits. Although clinical evidence indicates that revascularization of the ischemic brain regions is crucial for functional recovery, no therapeutics that promote angiogenesis after cerebral stroke are currently available. Besides vascular growth factors, guidance molecules have been identified to regulate aspects of angiogenesis in the central nervous system (CNS) and may provide targets for therapeutic angiogenesis. In this study, we demonstrate that genetic deletion of the neurite outgrowth inhibitor Nogo-A or one of its corresponding receptors, S1PR2, improves vascular sprouting and repair and reduces neurological deficits after cerebral ischemia in mice. These findings were reproduced in a therapeutic approach using intrathecal anti-Nogo-A antibodies; such a therapy is currently in clinical testing for spinal cord injury. These results provide a basis for a therapeutic blockage of inhibitory guidance molecules to improve vascular and neural repair after ischemic CNS injuries.
Asunto(s)
Anticuerpos Antiidiotipos/farmacología , Isquemia Encefálica/tratamiento farmacológico , Proteínas Nogo/genética , Receptores de Esfingosina-1-Fosfato/genética , Accidente Cerebrovascular/tratamiento farmacológico , Animales , Encéfalo/efectos de los fármacos , Encéfalo/patología , Isquemia Encefálica/genética , Isquemia Encefálica/inmunología , Isquemia Encefálica/patología , Sistema Nervioso Central/efectos de los fármacos , Sistema Nervioso Central/patología , Modelos Animales de Enfermedad , Humanos , Ratones , Neovascularización Fisiológica/genética , Neovascularización Fisiológica/inmunología , Neuronas/efectos de los fármacos , Neuronas/patología , Proteínas Nogo/antagonistas & inhibidores , Proteínas Nogo/inmunología , Tractos Piramidales/efectos de los fármacos , Tractos Piramidales/patología , Recuperación de la Función/genética , Receptores de Esfingosina-1-Fosfato/antagonistas & inhibidores , Receptores de Esfingosina-1-Fosfato/inmunología , Traumatismos de la Médula Espinal/tratamiento farmacológico , Traumatismos de la Médula Espinal/inmunología , Traumatismos de la Médula Espinal/patología , Accidente Cerebrovascular/genética , Accidente Cerebrovascular/inmunología , Accidente Cerebrovascular/patologíaRESUMEN
DEC-205 is a type I transmembrane multilectin receptor that is predominantly expressed on dendritic cells (DCs). Therefore, previous studies primarily focused on processing of DEC-205targeted antigens by this potent antigen presenting cell type. Here we show that Epstein-Barr virus (EBV) transformed lymphoblastoid B-cell lines (LCLs) not only express DEC-205 at similar levels to DCs, but also efficiently present targeted EBV nuclear antigen 1 (EBNA1) and EBV-latent membrane protein 1 (LMP1) to EBNA1- and LMP1-specific CD4+ and CD8+ T-cell clones in vitro. Targeting of antigens to DEC-205 on B cells led to more efficient MHC class II than I loading, and stimulated T cells more efficiently than targeting to DEC-205 on DCs. Although LCLs internalized DEC-205targeted antigens less efficiently than DCs, they retained them for longer time periods and delivered them to endosomal compartments that receive also B-cell receptor targeted proteins. This could facilitate prolonged T-cell stimulation and efficient MHC class II loading, and, indeed, CD4+ T-cell expansion by DEC-205targeted vaccination was significantly compromised in B-cell deficient mice. These studies suggest that B cells, activated by virus transformation or other means, can contribute to T-cell stimulation after DEC-205 targeting of antigens during vaccination.
Asunto(s)
Antígenos CD/metabolismo , Linfocitos B/inmunología , Linfocitos B/virología , Células Dendríticas/inmunología , Herpesvirus Humano 4/fisiología , Lectinas Tipo C/metabolismo , Receptores de Superficie Celular/metabolismo , Linfocitos T/inmunología , Animales , Presentación de Antígeno/fisiología , Antígenos CD/inmunología , Linfocitos B/metabolismo , Línea Celular Transformada , Transformación Celular Viral/inmunología , Células Cultivadas , Células Dendríticas/fisiología , Herpesvirus Humano 4/inmunología , Lectinas Tipo C/inmunología , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Antígenos de Histocompatibilidad Menor , Terapia Molecular Dirigida , Receptores de Antígenos de Linfocitos B/inmunología , Receptores de Antígenos de Linfocitos B/metabolismo , Receptores de Superficie Celular/inmunología , Linfocitos T/metabolismo , Linfocitos T/fisiología , Vacunación/métodosRESUMEN
Nogo-A is a transmembrane protein with multiple functions in the central nervous system (CNS), including restriction of neurite growth and synaptic plasticity. Thus far, Nogo-A has been predominantly considered a cell contact-dependent ligand signaling via cell surface receptors. Here, we show that Nogo-A can be secreted by cultured cells of neuronal and glial origin in association with extracellular vesicles (EVs). Neuron- and oligodendrocyte-derived Nogo-A containing EVs inhibited fibroblast spreading, and this effect was partially reversed by Nogo-A receptor S1PR2 blockage. EVs purified from HEK cells only inhibited fibroblast spreading upon Nogo-A over-expression. Nogo-A-containing EVs were found in vivo in the blood of healthy mice and rats, as well as in human plasma. Blood Nogo-A concentrations were elevated after acute stroke lesions in mice and rats. Nogo-A active peptides decreased barrier integrity in an in vitro blood-brain barrier model. Stroked mice showed increased dye permeability in peripheral organs when tested 2 weeks after injury. In the Miles assay, an in vivo test to assess leakage of the skin vasculature, a Nogo-A active peptide increased dye permeability. These findings suggest that blood borne, possibly EV-associated Nogo-A could exert long-range regulatory actions on vascular permeability.
RESUMEN
Lymph node-type T- and B-cell infiltrates with germinal centers are characteristic features of the hyperplastic thymus in early onset myasthenia gravis (EOMG).Epstein-Barr virus (EBV) infection confers survival advantages on B cells, and has recently been implicated in tertiary lymphoid tissue formation in EOMG. We evaluated the frequency of intrathymic EBV-infected B-lineage cells and antiviral immune responses in treatment-naive patients with EOMG. Real-time polymerase chain reaction was performed to quantify the content of genomic EBV DNA (BamHI-W repeat region) in thymic cell suspensions. Serial paraffin sections of EOMG thymi were analyzed for the presence of EBV-encoded RNA by in situ hybridization and for viral gene expression by immunohistochemistry. Humoral and cellular immune responses to viral antigens were quantified by enzyme-linked immunosorbent assay and flow cytometry-based intracellular cytokine staining. We detected minimal levels of viral DNA-corresponding to single viral genomes-in only 6 of 16 hyperplastic EOMG thymi, indicating extreme rarity of viral copy numbers in the investigated thymic samples. That was confirmed by similar rarity of EBV-encoded RNA and viral proteins identified in thymic sections. Furthermore, EBV-specific T- and B-cell responses were unchanged in patients with EOMG. These findings do not support an etiologic role for EBV in the initiation of EOMG.
Asunto(s)
Infecciones por Virus de Epstein-Barr/virología , Miastenia Gravis/virología , Timo/virología , Adolescente , Corticoesteroides/uso terapéutico , Adulto , Edad de Inicio , Antígenos CD19/metabolismo , Linfocitos B/fisiología , Linaje de la Célula/fisiología , Citocinas/metabolismo , ADN Viral/química , Ensayo de Inmunoadsorción Enzimática , Infecciones por Virus de Epstein-Barr/patología , Femenino , Citometría de Flujo , Humanos , Hiperplasia/patología , Inmunohistoquímica , Masculino , Miastenia Gravis/patología , Plásmidos/genética , Receptores Colinérgicos/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Timectomía , Timo/patología , Miembro 7 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/metabolismo , Adulto JovenRESUMEN
Blood brain barrier (BBB) damage is an important pathophysiological feature of ischemic stroke which significantly contributes to development of severe brain injury and therefore is an interesting target for therapeutic intervention. A popular permanent occlusion model to study long term recovery following stroke is the photothrombotic model, which so far has not been anatomically characterized for BBB leakage beyond the acute phase. Here, we observed enhanced BBB permeability over a time course of 3 weeks in peri-infarct and core regions of the ischemic cortex. Slight increases in BBB permeability could also be seen in the contralesional cortex, hippocampus and the cerebellum at different time points, regions where lesion-induced degeneration of pathways is prominent. Severe damage of tight and adherens junctions and loss of pericytes was observed within the peri-infarct region. Overall, the photothrombotic stroke model reproduces a variety of features observed in human stroke and thus, represents a suitable model to study BBB damage and therapeutic approaches interfering with this process.
RESUMEN
Angiogenesis is a key restorative process following stroke but has also been linked to increased vascular permeability and blood brain barrier (BBB) disruption. Previous pre-clinical approaches primarily focused on the administration of vascular endothelial growth factor (VEGF) to promote vascular repair after stroke. Although shown to improve angiogenesis and functional recovery from stroke, VEGF increased the risk of blood brain barrier disruption and bleedings to such an extent that its clinical use is contraindicated. As an alternative strategy, antibodies against the neurite growth inhibitory factor Nogo-A have recently been shown to enhance vascular regeneration in the ischemic central nervous system (CNS); however, their effect on vascular permeability is unknown. Here, we demonstrate that antibody-mediated Nogo-A neutralization following stroke has strong pro-angiogenic effects but does not increase vascular permeability as opposed to VEGF. Moreover, VEGF-induced vascular permeability was partially prevented when VEGF was co-administered with anti-Nogo-A antibodies. This study may provide a novel therapeutic strategy for vascular repair and maturation in the ischemic brain.
Asunto(s)
Inductores de la Angiogénesis/inmunología , Autoanticuerpos/inmunología , Permeabilidad Capilar/inmunología , Proteínas Nogo/inmunología , Accidente Cerebrovascular/fisiopatología , Animales , Modelos Animales de Enfermedad , Humanos , Neovascularización Patológica , Factores de Crecimiento Endotelial Vascular/administración & dosificaciónRESUMEN
Immunoglobulin A (IgA) plays an important role in protecting our mucosal surfaces from viral infection, in maintaining a balance with the commensal bacterial flora, and in extending maternal immunity via breast feeding. Here, we report an additional innate immune effector function of human IgA molecules in that we demonstrate that the C-terminal tail unique to IgA molecules interferes with cell-surface attachment of influenza A and other enveloped viruses that use sialic acid as a receptor. This antiviral activity is mediated by sialic acid found in the complex N-linked glycans at position 459. Antiviral activity was observed even in the absence of classical antibody binding via the antigen binding sites. Our data, therefore, show that the C-terminal tail of IgA subtypes provides an innate line of defense against viruses that use sialic acid as a receptor and the role of neuraminidases present on these virions.
Asunto(s)
Inmunidad Innata , Inmunoglobulina A/inmunología , Virus de la Influenza A/inmunología , Ácido N-Acetilneuramínico/metabolismo , Procesamiento Proteico-Postraduccional , Animales , Embrión de Pollo , Chlorocebus aethiops , Perros , Glicosilación , Células HEK293 , Humanos , Inmunoglobulina A/metabolismo , Células de Riñón Canino Madin Darby , Unión Proteica , Células VeroRESUMEN
Rituximab, a monoclonal B-cell cytolytic antibody, has beneficial effects in patients with inflammatory demyelinating diseases. So far, little data exists on B-cell subset recovery after rituximab treatment in inflammatory demyelinating diseases of the central nervous system (CNS). To elucidate whether rituximab promotes qualitative changes in the IgG memory B-cell repertoire we performed a single cell analysis in three patients with CNS demyelination. We did not observe any qualitative changes but detected an increased clonal expansion in the IgG memory B-cell compartment after treatment, indicating that a single course of rituximab does not eliminate specific IgG memory B-cells.
Asunto(s)
Linfocitos B/inmunología , Enfermedades Autoinmunes Desmielinizantes SNC/tratamiento farmacológico , Enfermedades Autoinmunes Desmielinizantes SNC/inmunología , Inmunoglobulina G/inmunología , Factores Inmunológicos/uso terapéutico , Rituximab/uso terapéutico , Linfocitos B/efectos de los fármacos , Femenino , Humanos , Factores Inmunológicos/farmacología , Masculino , Persona de Mediana Edad , Esclerosis Múltiple/tratamiento farmacológico , Esclerosis Múltiple/inmunología , Rituximab/farmacología , Adulto JovenRESUMEN
IgG molecules exert both pro- and antiinflammatory effector functions based on the composition of the fragment crystallizable (Fc) domain glycan. Sialylated IgG Fc domains have antiinflammatory properties that are attributed to their ability to increase the activation threshold of innate effector cells to immune complexes by stimulating the upregulation of the inhibitory Fcγ receptor IIB (FcγRIIB). Here, we report that IgG Fc sialylation of human monoclonal IgG1 molecules impairs their efficacy to induce complement-mediated cytotoxicity (CDC). Fc sialylation of a CD20-targeting antibody had no impact on antibody-dependent cellular cytotoxicity and did not change the affinity of the antibody for activating Fcγ receptors. In contrast, the presence of sialic acid abrogated the increased binding of C1q to Fc-galactosylated IgG1 and resulted in decreased levels of C3b deposition on the cell surface. Similar to monoclonal antibodies, sialic acid inhibited the increased C1q binding to galactosylated Fc fragments in human polyclonal IgG. In sera derived from patients with chronic inflammatory demyelinating polyneuropathy, an autoimmune disease of the peripheral nervous system in which humoral immune responses mediate tissue damage, induction of IgG Fc sialylation was associated with clinical disease remission. Thus, impairment of CDC represents an FcγR-independent mechanism by which Fc-sialylated glycovariants might limit proinflammatory IgG effector functions.
Asunto(s)
Linfocitos B/inmunología , Vía Clásica del Complemento , Proteínas del Sistema Complemento/inmunología , Inmunoglobulina G/química , Cadenas gamma de Inmunoglobulina/química , Ácido N-Acetilneuramínico/química , Rituximab/química , Animales , Citotoxicidad Celular Dependiente de Anticuerpos , Antígenos CD20/inmunología , Linfoma de Burkitt/patología , Línea Celular Tumoral , Complemento C1q/inmunología , Complemento C1q/metabolismo , Citotoxicidad Inmunológica , Glicosilación , Humanos , Inmunoglobulina G/inmunología , Cadenas gamma de Inmunoglobulina/inmunología , Inmunoglobulinas Intravenosas/uso terapéutico , Células Asesinas Naturales/inmunología , Depleción Linfocítica , Ratones , Glicoproteína Mielina-Oligodendrócito/inmunología , Polirradiculoneuropatía Crónica Inflamatoria Desmielinizante/inmunología , Polirradiculoneuropatía Crónica Inflamatoria Desmielinizante/terapia , Procesamiento Proteico-Postraduccional , Receptores de IgG/inmunología , Rituximab/inmunologíaRESUMEN
The B cell-depleting IgG1 monoclonal antibody rituximab can persistently suppress disease progression in some patients with autoimmune diseases. However, the mechanism underlying these long-term beneficial effects has remained unclear. Here, we evaluated Ig gene usage in patients with anti-myelin-associated glycoprotein (anti-MAG) neuropathy, an autoimmune disease of the peripheral nervous system that is mediated by IgM autoantibodies binding to MAG antigen. Patients with anti-MAG neuropathy showed substantial clonal expansions of blood IgM memory B cells that recognized MAG antigen. The group of patients showing no clinical improvement after rituximab therapy were distinguished from clinical responders by a higher load of clonal IgM memory B cell expansions before and after therapy, by persistence of clonal expansions despite efficient peripheral B cell depletion, and by a lack of substantial changes in somatic hypermutation frequencies of IgM memory B cells. We infer from these data that the effectiveness of rituximab therapy depends on efficient depletion of noncirculating B cells and is associated with qualitative immunological changes that indicate reconfiguration of B cell memory through sustained reduction of autoreactive clonal expansions. These findings support the continued development of B cell-depleting therapies for autoimmune diseases.