BackgroundVaccination strategies that elicit antigen-specific tolerance are needed as therapies for autoimmune disease. This study focused on whether cytokine-neuroantigen (NAg) fusion proteins could inhibit disease in chronic murine models of experimental autoimmune encephalomyelitis (EAE) and thus serve as potential therapeutic modalities for multiple sclerosis.ResultsA fusion protein comprised of murine GM-CSF as the N-terminal domain and the encephalitogenic MOG35-55 peptide as the C-terminal domain was tested as a tolerogenic, therapeutic vaccine (TTV) in the C57BL/6 model of EAE. Administration of GMCSF-MOG before active induction of EAE, or alternatively, at the onset of EAE blocked the development and progression of EAE. Covalent linkage of the GM-CSF and MOG35-55 domains was required for tolerogenic activity. Likewise, a TTV comprised of GM-CSF and PLP139-151 was a tolerogen in the SJL model of EAE.ConclusionThese data indicated that fusion proteins containing GM-CSF coupled to myelin auto-antigens elicit tolerance rather than immunity.

Irr and RirA, rather than Fur, serve as the major iron-responsive regulators in the alphaproteobacteria. With only a few exceptions, however, the relative contributions of these transcriptional regulators to the differential expression of specific iron metabolism genes in Brucella strains are unclear. The gene encoding the outer membrane heme transporter BhuA exhibits maximum expression in Brucella abortus 2308 during growth under iron-deprived conditions, and mutational studies indicate that this pattern of bhuA expression is mediated by the iron-responsive regulator Irr. Specifically, a bhuA-lacZ transcriptional fusion does not produce elevated levels of ␤-galactosidase in response to iron deprivation in the isogenic irr mutant BEA5, and, unlike the parental strain, B. abortus BEA5 cannot utilize heme as an iron source in vitro and is attenuated in mice. A derivative of the bhuA-lacZ transcriptional fusion lacking the predicted Irr binding site upstream of the bhuA promoter does not produce elevated levels of ␤-galactosidase in response to iron deprivation in the parental B. abortus 2308 strain, and a direct and specific interaction between a recombinant version of the Brucella Irr and the bhuA promoter region was observed in an electrophoretic mobility shift assay. Despite the fact that it lacks the heme regulatory element linked to the iron-responsive degradation of its counterpart in Bradyrhizobium japonicum, readily detectable levels of Irr were found only in B. abortus 2308 cells by Western blot analysis following growth under iron-deprived conditions.

The Brucella BhuQ protein is a homolog of the Bradyrhizobium japonicum heme oxygenases HmuD and HmuQ. To determine if this protein plays a role in the ability of Brucella abortus 2308 to use heme as an iron source, an isogenic bhuQ mutant was constructed and its phenotype evaluated. Although the Brucella abortus bhuQ mutant DCO1 did not exhibit a defect in its capacity to use heme as an iron source or evidence of increased heme toxicity in vitro, this mutant produced increased levels of siderophore in response to iron deprivation compared to 2308. Introduction of a bhuQ mutation into the B. abortus dhbC mutant BHB2 (which cannot produce siderophores) resulted in a severe growth defect in the dhbC bhuQ double mutant JFO1 during cultivation under iron-restricted conditions, which could be rescued by the addition of FeCl 3 , but not heme, to the growth medium. The bhuQ gene is cotranscribed with the gene encoding the iron-responsive regulator RirA, and both of these genes are repressed by the other major iron-responsive regulator in the alphaproteobacteria, Irr. The results of these studies suggest that B. abortus 2308 has at least one other heme oxygenase that works in concert with BhuQ to allow this strain to efficiently use heme as an iron source. The genetic organization of the rirA-bhuQ operon also provides the basis for the proposition that BhuQ may perform a previously unrecognized function by allowing the transcriptional regulator RirA to recognize heme as an iron source. Iron represents an essential micronutrient for Brucella strains (27). Acquiring sufficient iron to meet their physiological needs is particularly challenging for the brucellae because these bacteria are found in nature almost exclusively in mammalian hosts, an environment where the iron restriction faced by pathogenic microbes is well documented (29). Brucella strains can use heme as an iron source in vitro, and studies with an isogenic mutant have shown that the presence of the TonB-dependent outer membrane heme transporter BhuA is required for the wild-type virulence of B. abortus 2308 in experimentally infected mice (21), suggesting that heme is a biologically relevant source of iron for the brucellae during infection.Heme oxygenases catalyze the release of iron from heme, and these enzymes contribute to the ability of a variety of bacteria to utilize heme as an iron source (25,30,37). The product of the gene designated BMEII0706 in the Brucella melitensis 16M genome sequence shares 58 and 50% amino acid identity with the heme oxygenases HmuD and HmuQ, respectively, from Bradyrhizobium japonicum, and this Brucella protein exhibits heme oxygenase activity in an in vitro assay (23). Based on its documented biochemical activity and its homology to the IsdG/HmuQ family of heme oxygenases, we have given this protein the designation BhuQ (Brucella heme utilization oxygenase Q). The purpose of the experiments described in this report was to determine if the homologous protein in Brucella abortus 2308 (which is encoded by BAB2_0677) plays a rol...