Curcumin activates the p38MPAK-HSP25 pathway in vitro but fails to attenuate diabetic nephropathy in DBA2J mice despite urinary clearance documented by HPLC

<p>Abstract</p> <p>Background</p> <p>Curcumin has anti-inflammatory, anti-oxidant, and anti-proliferative properties, and depending upon the experimental circumstances, may be pro- or anti-apoptotic. Many of these biological actions could ameliorate diabetic nephropathy.</p> <p>Methods/Design</p> <p>Mouse podocytes, cultured in basal or high glucose conditions, underwent acute exposure to curcumin. Western blots for p38-MAPK, COX-2 and cleaved caspase-3; isoelectric focusing for HSP25 phosphorylation; and DNase I assays for F- to G- actin cleavage were performed for <it>in vitro </it>analyses. <it>In vivo </it>studies examined the effects of dietary curcumin on the development of diabetic nephropathy in streptozotocin (Stz)-induced diabetes in DBA2J mice. Urinary albumin to creatinine ratios were obtained, high performance liquid chromatography was performed for urinary curcuminoid measurements, and Western blots for p38-MAPK and total HSP25 were performed.</p> <p>Results</p> <p>Curcumin enhanced the phosphorylation of both p38MAPK and downstream HSP25; inhibited COX-2; induced a trend towards attenuation of F- to G-actin cleavage; and dramatically inhibited the activation of caspase-3 in <it>vitro</it>. In curcumin-treated DBA2J mice with Stz-diabetes, HPLC measurements confirmed the presence of urinary curcuminoid. Nevertheless, dietary provision of curcumin either before or after the induction of diabetes failed to attenuate albuminuria.</p> <p>Conclusions</p> <p>Apart from species, strain, early differences in glycemic control, and/or dosing effects, the failure to modulate albuminuria may have been due to a decrement in renal HSP25 or stimulation of the 12/15 lipoxygenase pathway in DBA2J mice fed curcumin. In addition, these studies suggest that timed urine collections may be useful for monitoring curcumin dosing and renal pharmacodynamic effects.</p

Dynamics and magnitude of virus-induced polyclonal B cell activation mediated by BCR-independent presentation of viral antigen

Hypergammaglobulinemia and production of autoantibodies occur during many viral infections, and studies have suggested that viral antigen-presenting B cells may become polyclonally activated by CD4 T cells in vivo in the absence of viral engagement of the BCR. However, we have reported that CD4 cells in lymphocytic choriomengitis virus (LCMV)-infected mice kill adoptively transferred B cells coated with LCMV class II peptides. We report here that most of the surviving naive B cells presenting class II MHC peptides undergo an extensive differentiation process involving both proliferation and secretion of antibodies. Both events require CD4 cells and CD40/CD40L interactions but not MyD88-dependent signaling within the B cells. B cells taken from immunologically tolerant donor LCMV-carrier mice with high LCMV antigen load became activated following adoptive transfer into LCMV-infected hosts, suggesting that B cells present sufficient antigen for this process during a viral infection. No division or activation of B cells was detected at all in virus-infected hosts in the absence of cognate CD4 T cells and class II antigen. This approach, therefore, formally demonstrates and quantifies a virus-induced polyclonal proliferation and differentiation of B cells, which, due to their high proportion, would mostly have BCR not specific for the virus

Immunogenicity of Influenza Virus Vaccine Is Increased by Anti-Gal-Mediated Targeting to Antigen-Presenting Cells▿

This study describes a method for increasing the immunogenicity of influenza virus vaccines by exploiting the natural anti-Gal antibody to effectively target vaccines to antigen-presenting cells (APC). This method is based on enzymatic engineering of carbohydrate chains on virus envelope hemagglutinin to carry the α-Gal epitope (Galα1-3Galβ1-4GlcNAc-R). This epitope interacts with anti-Gal, the most abundant antibody in humans (1% of immunoglobulins). Influenza virus vaccine expressing α-Gal epitopes is opsonized in situ by anti-Gal immunoglobulin G. The Fc portion of opsonizing anti-Gal interacts with Fcγ receptors on APC and induces effective uptake of the vaccine virus by APC. APC internalizes the opsonized virus to transport it to draining lymph nodes for stimulation of influenza virus-specific T cells, thereby eliciting a protective immune response. The efficacy of such an influenza vaccine was demonstrated in α1,3galactosyltransferase (α1,3GT) knockout mice, which produce anti-Gal, using the influenza virus strain A/Puerto Rico/8/34-H1N1 (PR8). Synthesis of α-Gal epitopes on carbohydrate chains of PR8 virus (PR8αgal) was catalyzed by recombinant α1,3GT, the glycosylation enzyme that synthesizes α-Gal epitopes in cells of nonprimate mammals. Mice immunized with PR8αgal displayed much higher numbers of PR8-specific CD8+ and CD4+ T cells (determined by intracellular cytokine staining and enzyme-linked immunospot assay) and produced anti-PR8 antibodies with much higher titers than mice immunized with PR8 lacking α-Gal epitopes. Mice immunized with PR8αgal also displayed a much higher level of protection than PR8 immunized mice after being challenged with lethal doses of live PR8 virus. We suggest that a similar method for increasing immunogenicity may be applicable to avian influenza vaccines

Generation of protective T cell-independent antiviral antibody responses in SCID mice reconstituted with follicular or marginal zone B cells

B cells generated in the bone marrow of adult mice enter the periphery as transitional B cells and subsequently differentiate into one of two phenotypically and functionally distinct subsets, marginal zone (MZ) or follicular (Fo) B cells. Recent reports indicate, however, that in response to environmental cues, such as lymphopenia, mature Fo B cells can change to display phenotypic markers characteristic of MZ B cells. Previously, we found that splenic B cells transferred to SCID mice responded to polyoma virus (PyV) infection with T cell-independent (TI) IgM and IgG secretion, reducing the viral load and protecting mice from the lethal effect of the infection. The contribution of MZ and Fo B cell subsets to this antiviral TI-2 response, however, has not been addressed. In this study, we show that both sort-purified MZ and Fo B cells generate protective TI Ab responses to PyV infection when transferred into SCID mice. Moreover, the transferred Fo B cells in the spleens of the PyV-infected SCID mice change phenotype, with many of them displaying MZ B cell characteristics. These findings demonstrate the plasticity of the B cell subsets in virus-infected hosts and show for the first time that B cells derived exclusively from Fo B cells can effectively function in antiviral TI-2 responses

MyD88 is required for the formation of long-term humoral immunity to virus infection

Development of long-term humoral immunity is a major goal of vaccination, but the mechanisms involved in the formation of long-term Ab responses are still being determined. In this study, we identify a previously unknown requirement for MyD88, an adaptor molecule that mediates signals at most TLRs, for the generation of long-term humoral immunity during live virus infection. Polyoma virus-infected MyD88 knockout mice generated strong acute T cell-dependent antiviral IgM and IgG responses and developed germinal centers. Activation-induced cytidine deaminase, an enzyme required for isotype switching and somatic hypermutation, was also induced in germinal center B cells, similar to wild-type mice. However, MyD88 knockout mice failed to develop bone marrow plasma cells and did not maintain long-term serum antiviral Ab responses. The isotype distribution of antiviral IgG responses was also altered; serum IgG2a and IgG2b levels were diminished, whereas IgG1 responses were not affected. The requirement for MyD88 for the formation of long-term humoral immunity to polyoma virus was intrinsic to B cells and was independent of IL-1R and IL-18R, cytokine receptors that also signal through MyD88. Our findings show that MyD88-dependent signaling pathways in B cells are essential for effectively generating long-term Ab responses and implicate a role for TLR in the formation of long-term humoral immunity