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Cholera toxin B subunit fusion to autoantigens such as proinsulin (CTB-INS) down regulate dendritic cell (DC) activation and stimulate synthesis of DC immunosuppressive cytokines. Recent studies of CTB-INS induction of immune tolerance in human DCs indicate that increased biosynthesis of indoleamine 2,3-dioxygenase (IDO1) may play an important role in CTB-INS vaccine suppression of DC activation. Studies in murine models suggest a role for transforming growth factor beta (TGF-β) in the stimulation of IDO1 biosynthesis, for the induction of tolerance in DCs. Here, we investigated the contribution of TGF-β superfamily proteins to CTB-INS induction of IDO1 biosynthesis in human monocyte-derived DCs (moDCs). We show that CTB-INS upregulates the level of TGF-β1, activin-A and the TGF-β activator, integrin αvβ8 in human DCs. However, inhibition of endogenous TGF-β, activin-A or addition of biologically active TGF-β1, and activin-A, did not inhibit or stimulate IDO1 biosynthesis in human DCs treated with CTB-INS. While inhibition with the kinase inhibitor, RepSox, blocked SMAD2/3 phosphorylation and diminished IDO1 biosynthesis in a concentration dependent manner. Specific blocking of the TGF-β type 1 kinase receptor with SB-431542 did not arrest IDO1 biosynthesis, suggesting the involvement of a different kinase pathway other than TGF-β type 1 receptor kinase in CTB-INS induction of IDO1 in human moDCs. Together, our experimental findings identify additional immunoregulatory proteins induced by the CTB-INS fusion protein, suggesting CTB-INS may utilize multiple mechanisms in the induction of tolerance in human moDCs. [ABSTRACT FROM AUTHOR]

Over the last 2 decades, the number of emergent infectious diseases has increased at an alarming rate. Also disheartening is the rise of known infectious pathogens that have acquired extensive drug resistance and reemerged with greater virulence. More recently, the threat of bioweapons has rekindled an urgency for the development of mass immunization programs. In response to this increased infectious disease threat, efforts have been intensified to identify more effective, inexpensive, and more easily deliverable mucosal vaccination methods. One area of research currently under development is the genetic modification of plants for production of immunoprotective proteins. The ability of plants to synthesize complex proteins using the elements of sunlight, soil, air, and water makes them ideal organisms for harvesting large quantities of therapeutic proteins. The introduction of antigen or antibody encoding genes into the genome of a plant through stable transformation enables them to manufacture vaccine proteins that are directly applicable for use in disease treatment, unlike yeast, bacterial, insect or other expression systems that require purification steps before delivery. As an alternative to stable transformation, plants can be used to generate large quantities of vaccines by acting as hosts for genetically altered plant viruses in which antigen proteins can be expressed and later purified from infected plant tissues. In this review, we survey current experimental strategies for using edible plants to achieve passive and active immunization against infectious disease organisms. In addition, methods are described for the construction of transformed plants that can provide protection against autoimmune diseases. Concerns and present obstacles to effective immunization with plant-based vaccines for animals and humans are presented. [ABSTRACT FROM AUTHOR]

An abstract of the article "Oral Delivery of Recombinant Vaccinia Viruses Expressing Adjuvanted Islet Autoantigens Protects NOD Mice from Autoimmune Diabetes," by William H. R. Langridge, Bela Denes, Nadia Fodor, John Hough, Jie Yu and Istvan Fodor is presented.

Abstract: A fusion protein containing the shiga toxin-1 B subunit (STB) linked to a 90 amino acid peptide (aa residues 86–175) from simian rotavirus (SA-11) nonstructural protein NSP4 was synthesized in Escherichia coli. Mice orally inoculated with 60μg of STB::NSP490 fusion protein per dose generated higher humoral and intestinal antibody titers than mice inoculated with 30μg of NSP4 alone. Serum anti-NSP4 IgG2a isotype titers were substantially greater than IgG1 titers, suggesting a dominant Th1 immune response. ELISA measurement of cytokines secreted from splenocytes isolated from immunized mice confirmed the STB::NSP490 fusion protein stimulation of a strong Th1 cell mediated immune response. Diarrhea in SA-11 rotavirus challenged neonates suckling from STB::NSP4 immunized dams was significantly reduced in severity and duration in comparison with virus challenged neonates from unimmunized mice. Together, our experiments demonstrate for the first time that the shiga toxin B subunit provides ligand mediated delivery of virus antigens to the gut-associated lymphoid tissues for enhanced stimulation of humoral and cellular responses against rotavirus gastroenteritis. [Copyright &y& Elsevier]

A cDNA fragment encoding the V3 loop of human immunodeficiency virus type-1 (HIV-1) envelope glycoprotein gp120 was fused to the cholera toxin B subunit gene (CTB–gp120) and transferred into Solanum tuberosum cells by Agrobacterium tumefaciens-mediated transformation. The CTB–gp120 fusion gene was detected in genomic DNA from transformed potato leaves by PCR DNA amplification. Synthesis and assembly of the CTB–gp120 fusion protein into oligomeric structures of pentamer size was detected in transformed tuber extracts by immunoblot analysis. The binding of CTB–gp120 fusion protein pentamers to intestinal epithelial cell membrane glycolipid receptors was quantified by GM1-ganglioside enzyme-linked immunosorbent assay (GM1-ELISA). The ELISA results indicated that CTB–gp120 fusion protein made up 0.002–0.004% of the total soluble tuber protein. Synthesis of CTB–gp120 monomers and their assembly into biologically active oligomers in transformed potato tuber tissues demonstrates for the first time the expression of HIV-1 gp120 in plants and emphasizes the feasibility of using edible plant-based vaccination for protection against HIV-1 infection. [Copyright &y& Elsevier]

A cDNA encoding the Simian immunodeficiency virus type (SIVmac) Gag capsid protein was introduced into Solanum tuberosum cells by Agrobacterium tumefaciens-mediated transformation methods. The gag gene was detected in the genomic DNA of transformed leaf tissues by PCR DNA amplification. Immunoblot analysis of transformed potato plant extracts with anti-Gag monoclonal antibody showed that biologically active Gag protein was synthesized in transformed tuber tissues. Based on ELISA results, recombinant Gag protein made up 0.006–0.014% of total soluble tuber protein. The synthesis of SIV Gag in transformed potato tubers opens the way for development of Gag-based edible plant vaccines for protection against SIV and potentially HIV-1 infection. [Copyright &y& Elsevier]

A cDNA encoding the simian–human immunodeficiency virus (SHIV 89.6p) Tat regulatory element protein was fused to the c-terminus of the cholera toxin B subunit gene (ctxB-tat) and introduced into Solanum tuberosum cells by Agrobacterium tumefaciens-mediated transformation methods. The fusion gene was detected in the genomic DNA of transformed potato leaf cells by PCR DNA amplification. Synthesis and assembly of the CTB-Tat fusion protein into oligomeric structures of pentamer size was detected in transformed tuber extracts by immunoblot analysis. The binding of CTB-Tat fusion protein pentamers to intestinal epithelial cell membrane glycolipid receptors was quantified by GM1-ganglioside enzyme-linked immunosorbent assay (GM1-ELISA). Based on the ELISA results, CTB-Tat fusion protein made up about 0.005–0.007% of total soluble tuber protein or approximately 4.6 mg per 100 g potato tuber tissue. The synthesis and assembly of CTB-Tat monomers into biologically active oligomers in transformed potato tuber tissues demonstrates the feasibility of using viral pathogen antigens synthesized in edible plants for mucosal immunization against HIV-1 infection. [Copyright &y& Elsevier]

Graphical abstract Highlights • Extracellular vimentin decreases secretion of IL6 and IL12 in dendritic cells (DCs). • Vimentin does not change DC surface expression of MHC, CD80, CD86, or CD83. • Extracellular vimentin decreases DC-mediated T cell secretion of IFN-γ. Abstract Vimentin is an intermediate filament protein traditionally considered to be an intracellular protein with a structural role. However, recent evidence suggests that vimentin can also be found outside the cell in disease conditions such as cancer, traumatic tissue injury, and inflammation. Extracellular vimentin was previously found to stimulate innate immunity by increasing monocyte and macrophage ability to kill bacteria. However, vimentin has also been previously found to decrease neutrophil infiltration into inflamed tissue. How extracellular vimentin affects the initiation of adaptive immune responses is unknown. Initiation of adaptive immunity involves priming of naïve T cells by antigen-presenting cells, the most effective of which are dendritic cells (DCs). In this study, we demonstrate how extracellular vimentin modulates lipopolysaccharide (LPS) – induced activation of human DCs. Using cytometric bead arrays, we show that extracellular vimentin decreases LPS-activated DC secretion of pro-inflammatory cytokines IL-6 and IL-12 while increasing secretion of the anti-inflammatory cytokine IL-10. Using flow cytometry, we show that extracellular vimentin does not significantly affect LPS-induced DC surface expression of MHC I (HLA-ABC) or MHC II (HLA-DR) presentation molecules, costimulatory factors (CD80, CD86), or the DC maturation marker (CD83). Further, LPS-stimulated DCs co-cultured with allogeneic naïve CD4 + T cells (Th0) induced less secretion of the pro-inflammatory Th1 effector cytokine IFN-γ in the presence of vimentin than in the presence of LPS alone. This result suggests that vimentin reduces Th1 differentiation. Taken together, our data suggest that extracellular vimentin may inhibit pro-inflammatory adaptive immune responses, by blocking DC secretion of pro-inflammatory cytokines. Thus, extracellular vimentin may play an important role in cancer or trauma-complications by inducing suppression of the adaptive immune response. In a positive sense, the presence of extracellular vimentin may prevent tissue-damage from contributing to the development of autoimmunity. Consequently, extracellular vimentin may become a novel drug target for treatment of a variety of pro- and anti-inflammatory disease conditions. [ABSTRACT FROM AUTHOR]

Abstract: The gram-negative anaerobic oral bacterium Porphyromonas gingivalis initiates periodontal disease by binding to saliva-coated oral surfaces. To assess whether edible plants can synthesize biologically active P. gingivalis fimbrial antigen, for application as an oral vaccine, a cDNA fragment encoding the C-terminal binding portion of P. gingivalis fimbrial protein (FimA), was cloned into a plant expression vector immediately downstream of a cDNA fragment encoding the cholera toxin B subunit (CTB). The chimeric plasmid was transferred into potato (Solanum tuberosum) cells and the ctb–fimA cDNA fragment detected in transformed leaf genomic DNA by PCR amplification methods. A novel protein band of 21kDa was detected in transformed potato tuber extracts by immunoblot analysis. Oligomeric CTB–FimA (266–337) fusion protein was identified in the extracts through the binding of anti-CTX and anti-native fimbriae antibodies. The pentameric structure of CTB–FimA fusion protein was confirmed by ELISA measurements of GM1 ganglioside receptor binding. Quantification of the CTB–FimA fusion protein by ELISA indicated that the chimeric protein made up about 0.33% of total soluble tuber protein. The biosynthesis of immunologically detectable CTB–FimA fusion proteins and the assembly of fusion protein monomers into biologically active pentamers in transformed potato tuber tissues demonstrate the feasibility of synthesizing adjuvanted fimbrial protein in edible plants for development of adjuvanted mucosal vaccines against P. gingivalis generated periodontal disease. [Copyright &y& Elsevier]