RESUMEN
Shortage of oxygen and nutrients in the brain induces the release of glutamate and ATP that can cause excitotoxicity and contribute to neuronal and glial damage. Our understanding of the mechanisms of ATP release and toxicity in cerebrovascular diseases is incomplete. This review aims at summarizing current knowledge about the participation of key elements in the ATP-mediated deleterious effects in these pathologies. This includes pannexin-1 hemichannels, calcium homeostasis modulator-1 (CALHM1), purinergic P2X7 receptors, and other intermediaries of CNS injury downstream of ATP release. Available data together with recent pharmacological developments in purinergic signaling may constitute a new opportunity to translate preclinical findings into more effective therapies in cerebrovascular diseases.
RESUMEN
In addition to conventional antibodies, cartilaginous fish have evolved a distinctive type of immunoglobulin, designated as IgNAR, which lacks the light polypeptide chains and is composed entirely by heavy chains. IgNAR molecules can be manipulated by molecular engineering to produce the variable domain of a single heavy chain polypeptide (vNARs). These, together with the VHH camel domains, constitute the smallest naturally occurring domains able to recognize an antigen. Their special features, such as small size, long extended finger-like CDR3, and thermal and chemical stability, make them suitable candidates for biotechnological purposes. Here we describe the generation of two mouse monoclonal antibodies (MAbs), MAb 370-12 and MAb 533-10, that both specifically react with vNAR domains of the horn shark Heterodontus francisci. While the former recognizes a broad spectrum of recombinant vNAR proteins, the latter is more restricted. MAb 370-12 precipitated a single band from whole shark serum, which was identified as IgNAR by mass spectrometry. Additionally, we used MAb 370-12 to follow the IgNAR-mediated immune response of sharks during immunization protocols with two different antigens (complete cells and a synthethic peptide), thus corroborating that MAb 370-12 recognizes both isolated vNAR domains and whole IgNAR molecules. Both MAbs represent an affordable molecular, biochemical, and biotechnological tool in the field of shark single-domain antibodies.
Asunto(s)
Anticuerpos Monoclonales de Origen Murino/genética , Proteínas de Peces/genética , Región Variable de Inmunoglobulina/genética , Inmunoglobulinas/aislamiento & purificación , Tiburones/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales de Origen Murino/inmunología , Anticuerpos Monoclonales de Origen Murino/metabolismo , Especificidad de Anticuerpos , Células Cultivadas , Epítopos/química , Eritrocitos/inmunología , Proteínas de Peces/inmunología , Proteínas de Peces/metabolismo , Humanos , Hibridomas/metabolismo , Inmunidad Humoral , Región Variable de Inmunoglobulina/inmunología , Región Variable de Inmunoglobulina/metabolismo , Inmunoglobulinas/genética , Inmunoglobulinas/metabolismo , Inmunoprecipitación , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Unión Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismo , Tiburones/sangreRESUMEN
ADP-ribosylation of host cell proteins is a common mode of cell intoxication by pathogenic bacterial toxins. Antibodies induced by immunization with inactivated ADP-ribosylating toxins provide efficient protection in case of some secreted toxins, e.g., diphtheria and pertussis toxins. However, other ADP-ribosylating toxins, such as Salmonella SpvB toxin, are secreted directly from the Salmonella-containing vacuole into the cytosol of target cells via the SPI-2 encoded bacterial type III secretion system, and thus are inaccessible to conventional antibodies. Small-molecule ADP-ribosylation inhibitors are fraught with potential side effects caused by inhibition of endogenous ADP-ribosyltransferases. Here, we report the development of a single-domain antibody from an immunized llama that blocks the capacity of SpvB to ADP-ribosylate actin at a molar ratio of 1:1. The single-domain antibody, when expressed as an intrabody, effectively protected cells from the cytotoxic activity of a translocation-competent chimeric C2IN-C/SpvB toxin. Transfected cells were also protected against cytoskeletal alterations induced by wild-type SpvB-expressing strains of Salmonella. This proof of principle paves the way for developing new antidotes against intracellular toxins.