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1.
Front Immunol ; 9: 3139, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-30687332

RESUMEN

Co-ligation of the B cell antigen receptor with complement receptor 2 on B-cells via a C3d-opsonised antigen complex significantly lowers the threshold required for B cell activation. Consequently, fusions of antigens with C3d polymers have shown great potential in vaccine design. However, these linear arrays of C3d multimers do not mimic the natural opsonisation of antigens with C3d. Here we investigate the potential of using the unique complement activating characteristics of Staphylococcal immune-evasion protein Sbi to develop a pro-vaccine approach that spontaneously coats antigens with C3 degradation products in a natural way. We show that Sbi rapidly triggers the alternative complement pathway through recruitment of complement regulators, forming tripartite complexes that act as competitive antagonists of factor H, resulting in enhanced complement consumption. These functional results are corroborated by the structure of the complement activating Sbi-III-IV:C3d:FHR-1 complex. Finally, we demonstrate that Sbi, fused with Mycobacterium tuberculosis antigen Ag85b, causes efficient opsonisation with C3 fragments, thereby enhancing the immune response significantly beyond that of Ag85b alone, providing proof of concept for our pro-vaccine approach.


Asunto(s)
Adyuvantes Inmunológicos , Proteínas Bacterianas/inmunología , Proteínas Portadoras/inmunología , Evasión Inmune , Infecciones Estafilocócicas/inmunología , Vacunas Estafilocócicas/inmunología , Staphylococcus/inmunología , Aciltransferasas/genética , Aciltransferasas/inmunología , Adyuvantes Inmunológicos/química , Adyuvantes Inmunológicos/genética , Animales , Antígenos Bacterianos/genética , Antígenos Bacterianos/inmunología , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Portadoras/química , Proteínas Portadoras/genética , Modelos Animales de Enfermedad , Inmunización , Ratones , Ratones Noqueados , Modelos Moleculares , Conformación Proteica , Proteínas Recombinantes de Fusión/inmunología , Infecciones Estafilocócicas/prevención & control , Relación Estructura-Actividad
2.
Immunobiology ; 223(1): 125-134, 2018 01.
Artículo en Inglés | MEDLINE | ID: mdl-29017821

RESUMEN

The use of C3d, the final degradation product of complement protein C3, as a "natural" adjuvant has been widely examined since the initial documentation of its immunogenicity-enhancing properties as a consequence of binding to complement receptor 2. Subsequently it was demonstrated that these effects are most evident when oligomeric, rather than when monomeric forms of C3d, are linked to various test protein antigens. In this study, we examined the feasibility of enhancing the adjuvant properties of human C3d further by utilizing C4b-binding protein (C4BP) to provide an oligomeric arrayed scaffold fused to the model antigen, tetanus toxin C fragment (TTCF). High molecular weight, C3d-containing oligomeric vaccines were successfully expressed, purified from mammalian cells and used to immunize groups of mice. Surprisingly, anti-TTCF antibody responses measured in these mice were poor. Subsequently we established by in vitro and in vivo analysis that, in the presence of mouse C3, human C3d does not interact with either mouse or even human complement receptor 2. These data confirm the requirement to develop murine versions of C3d based adjuvant compounds to test in mice or that mice would need to be developed that express both human C3 and human CR2 to allow the testing of human C3d based adjuvants in mouse in any capacity.


Asunto(s)
Linfocitos B/fisiología , Complemento C3d/inmunología , Proteína de Unión al Complemento C4b/genética , Fragmentos de Péptidos/inmunología , Toxina Tetánica/inmunología , Vacunas Sintéticas/inmunología , Adyuvantes Inmunológicos , Animales , Anticuerpos/sangre , Línea Celular , Complemento C3d/genética , Proteína de Unión al Complemento C4b/inmunología , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Animales , Fragmentos de Péptidos/genética , Multimerización de Proteína/genética , Receptores de Complemento 3d/genética , Receptores de Complemento 3d/metabolismo , Toxina Tetánica/genética , Vacunación , Vacunas Sintéticas/genética
3.
Brain Struct Funct ; 222(8): 3567-3585, 2017 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-28386779

RESUMEN

Cortical interneurons are generated in the ganglionic eminences and migrate through the ventral and dorsal telencephalon before finding their final positions within the cortical plate. During early stages of migration, these cells are present in two well-defined streams within the developing cortex. In an attempt to identify candidate genes which may play a role in interneuron stream specification, we previously carried out a microarray analysis which identified a number of cadherin receptors that were differentially expressed in these streams, including Cadherin-13 (Cdh13). Expression analysis confirmed Cdh13 to be present in the preplate layer at E13.5 and, later in development, in some cortical interneurons and pyramidal cells. Analysis of Cdh13 knockout mice at E18.5, but not at E15.5, showed a reduction in the number of interneurons and late born pyramidal neurons and a concomitant increase in apoptotic cells in the cortex. These observations were confirmed in dissociated cell cultures using overexpression and short interfering RNAs (siRNAs) constructs and dominant negative inhibitory proteins. Our findings identified a novel protective role for Cdh13 in cortical neuron development.


Asunto(s)
Cadherinas/fisiología , Corteza Cerebral/embriología , Interneuronas/fisiología , Animales , Apoptosis , Cadherinas/genética , Cadherinas/metabolismo , Recuento de Células , Movimiento Celular , Proliferación Celular , Células Cultivadas , Corteza Cerebral/metabolismo , Femenino , Expresión Génica , Interneuronas/metabolismo , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo
4.
Neural Plast ; 2016: 6170509, 2016.
Artículo en Inglés | MEDLINE | ID: mdl-26881123

RESUMEN

The laying down of memory requires strong stimulation resulting in specific changes in synaptic strength and corresponding changes in size of dendritic spines. Strong stimuli can also be pathological, causing a homeostatic response, depressing and shrinking the synapse to prevent damage from too much Ca(2+) influx. But do all types of dendritic spines serve both of these apparently opposite functions? Using confocal microscopy in organotypic slices from mice expressing green fluorescent protein in hippocampal neurones, the size of individual spines along sections of dendrite has been tracked in response to application of tetraethylammonium. This strong stimulus would be expected to cause both a protective homeostatic response and long-term potentiation. We report separation of these functions, with spines of different sizes reacting differently to the same strong stimulus. The immediate shrinkage of large spines suggests a homeostatic protective response during the period of potential danger. In CA1, long-lasting growth of small spines subsequently occurs consolidating long-term potentiation but only after the large spines return to their original size. In contrast, small spines do not change in dentate gyrus where potentiation does not occur. The separation in time of these changes allows clear functional differentiation of spines of different sizes.


Asunto(s)
Espinas Dendríticas/fisiología , Hipocampo/citología , Hipocampo/fisiología , Homeostasis , Potenciación a Largo Plazo , Células Piramidales/citología , Células Piramidales/fisiología , Animales , Células Cultivadas , Espinas Dendríticas/efectos de los fármacos , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Femenino , Hipocampo/efectos de los fármacos , Potenciación a Largo Plazo/efectos de los fármacos , Masculino , Ratones , Células Piramidales/efectos de los fármacos , Tetraetilamonio/farmacología
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