Chemoattraction of leukocytes into the brain after induction of middle cerebral artery occlusion (MCAO) increases the lesion size and worsens disease outcome. Our previous studies demonstrated that partial MHC class II constructs can reverse this process. However, the potential application of pMHC to human stroke is limited by the need to rapidly match recipient MHC class II with the β1 domain of the pMHC construct. We designed a novel recombinant protein comprised of the HLA-DRα1 domain linked to MOG-35-55 peptide but lacking the β1 domain found in pMHC and treated MCAO after 4 h reperfusion in humanized DR2 mice. Infarct volumes were quantified after 96 h reperfusion and immune cells from the periphery and CNS were evaluated for expression of CD74 and other cell surface, cytokine and pathway markers. This study demonstrates that four daily treatments with DRα1-MOG-35-55 reduced infarct size by 40 % in the cortex, striatum and hemisphere, inhibited the migration of activated CD11b+CD45high cells from the periphery to the brain and reversed splenic atrophy. Furthermore, DRα1-MOG-35-55 bound to CD74 on monocytes and blocked both binding and downstream signaling of macrophage migration inhibition factor (MIF) that may play a key role in infarct development. The novel DRα1-MOG-35-55 construct is highly therapeutic in experimental stroke and could be given to all patients at least 4 h after stroke onset without the need for tissue typing due to universal expression of DRα1 in humans.

Recombinant T-cell Receptor Ligand 1000 (RTL1000), a partial human major histocompatibility complex (MHC) molecule coupled to a human myelin peptide, reduces infarct size after experimental stroke in HLA-DRB1*1502 transgenic (DR2-Tg) mice. In this study, we characterized the therapeutic time window of opportunity for RTL1000; we explored the efficacy of single dose of RTL1000 administration and determined if RTL1000 affordslong-term neurobehavioral functional improvement after ischemic stroke. Male DR2-Tg mice underwent 60 min of intraluminal reversible middle cerebral artery occlusion (MCAO). RTL1000 or vehicle was injected 4, 6 or 8 h after MCAO, followed by 3 daily injections. In single dose study, one-time injection of RTL1000 was applied 4 h after MCAO. Cortical, striatal and hemispheric infarct sizes were measured 24 h or 96 h after stroke. Behavioral testing, including neuroscore evaluation, open field, paw preference and novel object recognition was performed up to 28 days after stroke. Our data showed RTL1000 significantly reduced infarct size 96 h after MCAO when first injection was given 4 and 6, but not 8 h after the onset of stroke. A single dose of 400 µg or 100 µg RTL1000 also significantly reduced infarct size 24 h after MCAO. Behavioral testing showed RTL1000 treatment used 4 h after MCAO improved long-term cognitive outcome 28 days after stroke. Taken together, RTL1000 protects against acute injury if applied within a 6-h time window and improves long-term functional recovery after experimental stroke in DR2-Tg mice.

RTL1000 is a partial human MHC molecule coupled to a human myelin peptide. We previously demonstrated that RTL1000 was protective against experimental ischemic stroke in HLA-DR2 transgenic (DR2-Tg) mice. Since thrombolysis with recombinant tissue plasminogen activator (t-PA) is a standard therapy for stroke, we determined if RTL1000 efficacy is altered when combined with t-PA in experimental stroke. Male DR2-Tg mice underwent 60 min of intraluminal middle cerebral artery occlusion (MCAO). t-PA or vehicle was infused intravenously followed by either a single or 4 daily subcutaneous injections of RTL1000 or vehicle. Infarct size was measured by 2, 3, 5-triphenyltetrazolium chloride staining at 24h or 96 h of reperfusion. Our data showed that t-PA alone reduced infarct size when measured at 24 h but not at 96 h after MCAO. RTL1000 alone reduced infarct size both at 24 and 96h after MCAO. Combining RTL1000 with t-PA did not alter its ability to reduce infarct size at either 24 or 96 h after MCAO and provides additional protection in t-PA treated mice at 24 h after ischemic stroke. Taken together, RTL1000 treatment alone improves outcome and provides additional protection in t-PA treated mice in experimental ischemic stroke.

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The original version of this article unfortunately contained a mistake. In the Acknowledgements section, the funding source "#NS076013 (STTR)" is incorrect and should have been listed as "NS065515-02A1 (STTR)".The online version of the original article can be found at http://dx