DARC shuttles inflammatory chemokines across the blood-brain barrier during autoimmune central nervous system inflammation

The Duffy antigen/receptor for chemokines, DARC, belongs to the family of atypical heptahelical chemokine receptors that do not couple to G proteins and therefore fail to transmit conventional intracellular signals. Here we show that during experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis, the expression of DARC is upregulated at the blood-brain barrier. These findings are corroborated by the presence of a significantly increased number of subcortical white matter microvessels staining positive for DARC in human multiple sclerosis brains as compared to control tissue. Using an in vitro blood-brain barrier model we demonstrated that endothelial DARC mediates the abluminal to luminal transport of inflammatory chemokines across the blood-brain barrier. An involvement of DARC in experimental autoimmune encephalomyelitis pathogenesis was confirmed by the observed ameliorated experimental autoimmune encephalomyelitis in Darc(-/-) C57BL/6 and SJL mice, as compared to wild-type control littermates. Experimental autoimmune encephalomyelitis studies in bone marrow chimeric Darc(-/-) and wild-type mice revealed that increased plasma levels of inflammatory chemokines in experimental autoimmune encephalomyelitis depended on the presence of erythrocyte DARC. However, fully developed experimental autoimmune encephalomyelitis required the expression of endothelial DARC. Taken together, our data show a role for erythrocyte DARC as a chemokine reservoir and that endothelial DARC contributes to the pathogenesis of experimental autoimmune encephalomyelitis by shuttling chemokines across the blood-brain barrier.

Probing the effect of minor groove interactions on the catalytic efficiency of DNAzymes 8–17 and 10–23

DNAzymes (Dz) 8–17 and 10–23 are two widely studied and well-characterized RNA-cleaving DNA catalysts. In an effort to further improve the understanding of the fragile interactions and dynamics of the enzymatic mechanism, this study examines the catalytic efficiency of minimally modified DNAzymes. Five single mutants of Dz8–17 and Dz10–23 were prepared by replacing the adenine residues in the corresponding catalytic cores with 3-deazaadenine units. Kinetic assays were used to assess the effect on the catalytic activity and thereby identify the importance of hydrogen bonding that arises from the N3 atoms. The results suggest that modifications at A15 and A15.0 of Dz8–17 have a significant influence and show a reduction in catalytic activity. Modification at each location in Dz10–23 results in a decrease of the observed rate constants, with A12 appearing to be the most affected with a reduction of ∼80% of kobs and ∼25% of the maximal cleavage rate compared to the wild-type DNAzyme. On the other hand, modification of A12 in Dz8–17 showed an ∼130% increase in kobs, thus unraveling a new potential site for the introduction of chemical modifications. A pH-profile analysis showed that the chemical cleavage step is rate-determining, regardless of the presence and/or location of the mutation. These findings point towards the importance of the N3-nitrogens of certain adenine nucleotides located within the catalytic cores of the DNAzymes for efficient catalytic activity and further suggest that they might directly partake in maintaining the appropriate tertiary structure. Therefore, it appears that minor groove interactions constitute an important feature of DNAzymes as well as ribozymes.