Publications (152) View all
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Article: Re-utilization of germinal centers in multiple Peyer's patches results in highly synchronized, oligoclonal, and affinity-matured gut IgA responses.
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ABSTRACT: Whereas gut IgA responses to the microbiota may be multi-centered and diverse, little is known about IgA responses to T-cell-dependent antigens following oral immunizations. Using a novel approach, gut IgA responses to oral hapten (4-hydroxy-3-nitrophenyl)acetyl-cholera toxin (NP-CT) conjugates were followed at the cellular and molecular level. Surprisingly, these responses were highly synchronized, strongly oligoclonal, and dominated by affinity matured cells. Extensive lineage trees revealed clonal relationships between NP-specific IgA cells in gut inductive and effector sites, suggesting expansion of the same B-cell clone in multiple Peyer's patches (PPs). Adoptive transfer experiments showed that this was achieved through re-utilization of already existing germinal centers (GCs) in multiple PPs by previously activated GC GL7(+) B cells, provided oral NP-CT was given before cell transfer. Taken together, these results explain why repeated oral immunizations are mandatory for an effective oral vaccine.Mucosal Immunology advance online publication 11 July 2012. doi:10.1038/mi.2012.56.Mucosal Immunology 07/2012; · 6.96 Impact Factor -
Article: Gene expression profiling identifies STAT3 as a novel pathway for immunomodulation by cholera toxin adjuvant.
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ABSTRACT: Earlier studies have reported on both proinflammatory and anti-inflammatory activities of cholera toxin (CT). As CT is a powerful adjuvant, we were interested in identifying genes with a possible involvement in these functions. A global gene expression analysis in mouse B cells showed that CT regulated <100 annotated genes, which encoded transcription factors, G proteins, cell-cycle regulators, and immunoregulating molecules. Interestingly, CT regulated the expression of the signal transducer and activator of transcription (STAT)3 gene and influenced the level and activation of both isoforms STAT3 alpha and STAT3 beta, in vitro in a B-cell line and in Peyer's patch (PP) B cells and in vivo in freshly isolated splenic B cells from CT-treated mice. This effect was cAMP dependent and was not seen with CTB. B cells pre-exposed to CT were significantly more susceptible to the activation of STAT3 by interleukin (IL)-6 and IL-10. This exerted a stronger inhibitory effect of IL-10 on lipopolysaccharide (LPS)-stimulated B-cell proliferation and cytokine production (IL-6). Moreover, IgG1 and IgA production induced by LPS and IL-10 were enhanced by the addition of CT to cultures of PP or splenic B cells. This is the first study to provide a molecular mechanism that can reconcile previous findings of proinflammatory and anti-inflammatory effects by CT adjuvant.Mucosal Immunology 04/2010; 3(4):374-86. · 6.96 Impact Factor -
Article: Measurement of immunoglobulin synthesis using the ELISPOT assay.
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ABSTRACT: The enzyme-linked immunospot (ELISPOT) assay for detection of single antibody-secreting cells has become the best alternative method to the conventional plaque-forming cell (PFC) assays. Among its several advantages are better antigen stability and specificity as well as fewer limitations in the diversity of antigens that can be used in the assay. In addition, the ELISPOT assay can be used to detect two antigenically different secreted antibodies simultaneously by two-color analysis and offers the unique possibility of quantifying the number of antibody molecules secreted per cell. Finally, the assay can be used to detect single antibody-secreting cells in tissues that usually confront the immunologist with difficulties, e.g., gut lamina propria from humans or mice. This unit presents the ELISPOT assay in three steps: coating of antigen to a solid phase, incubation of antibody-producing cells in appropriate dilution, and detection of the antigen-antibody complex formed at the site of the active antibody-secreting cell. The assay can be performed using polystyrene plates or nitrocellulose membrane in microtiter plates or using nitrocellulose membrane in a blotting manifold.Current protocols in immunology / edited by John E. Coligan ... [et al.] 06/2001; Chapter 7:Unit 7.14. -
Article: Impaired CD40-signalling in CD19-deficient mice selectively affects Th2-dependent isotype switching.
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ABSTRACT: Activation of B lymphocytes involves binding of antigen to the specific receptor and signalling through several membrane coreceptors, of which CD19 has been found to play a pivotal role as a response regulator. Although previous studies in CD19 gene knockout mice have demonstrated that antibody responses to T-cell-dependent antigens are strongly impaired in the absence of this coreceptor, little is known about the consequences of CD19 deficiency for the interaction between T and B cells. Here we report that Th2 co-ordinated B-cell differentiation is selectively impaired in CD19-deficient mice in response to mucosal or systemic immunizations or following an intestinal infection with Nippostrongylus brasiliensis. Whereas immunoglobulin (Ig)G1 or IgE antibody responses were low or absent, IgG2a responses were normal. The selective defect was not caused by a poor Th2-development or interleukin (IL)-4 responsiveness in CD19-deficient mice. Rather, it was the result of an impaired Th2-B cell interaction, owing to a substantially reduced ability to signal via CD40 in CD19-deficient B cells. Thus, our study in CD19-deficient mice suggests that CD40L-CD40-interactions are more important for Th2 than for Th1 co-ordinated B-cell differentiation.Scandinavian Journal of Immunology 02/2001; 53(1):13-23. · 2.23 Impact Factor -
Article: CD19-deficient mice exhibit poor responsiveness to oral immunization despite evidence of unaltered total IgA levels, germinal centers and IgA-isotype switching in Peyer's patches.
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ABSTRACT: CD19 exhibits a critical role as a response regulator in B cells, influencing activation, differentiation and survival. Accordingly, CD19-deficient mice largely lack B-1 cells, and their conventional B-2 cells are poor responders to thymus-dependent antigen. Since both B-1 and B-2 cells may contribute to the total intestinal IgA production, we investigated whether lack of CD19 negatively affected mucosal immunity. We found that CD19(-/-) mice have near normal total IgA levels in serum and gut mucosa and, contrary to systemic lymphoid tissues, Peyer's patches (PP) had germinal centers to which also IgA+ B cells localized. However, the mice demonstrated severely impaired responses to oral immunization with keyhole limpet hemocyanin plus cholera toxin adjuvant. Mucosal responses to oral immunization were significantly more impaired than systemic responses. Despite normal specific IL-4 production, a selective defect in Th2-regulated B cell isotypes was observed, with poor or no mucosal IgA, low serum IgG1 and no IgE, but intact serum IgA and IgG2a production. Ex vivo experiments revealed strongly inhibited CD40-stimulated proliferation and IgA differentiation in CD19-deficient PP B cells. Taken together, an impaired CD40 responsiveness selectively affected Th2, but not Th1, coordinated B cell responses. The CD19(-/-) mice provide compelling evidence for the differential regulation of serum and mucosal IgA immunity.European Journal of Immunology 08/2000; 30(7):1861-71. · 5.10 Impact Factor
