Background: Inflammatory stimuli induce immunoresponsive gene 1 (IRG1) expression that in turn catalyzes the production of itaconate from the tricarboxylic acid cycle. Itaconate has recently emerged as a regulator of immune cell functions, especially in macrophages. Studies show that itaconate is required for the activation of anti-inflammatory transcription factor Nrf2 by LPS in mouse and human macrophages, and LPS-activated IRG1-/macrophages that lack endogenous itaconate production exhibit augmented inflammatory responses. Moreover, dimethyl itaconate (DMI), an itaconate derivative, inhibits IL-17-induced IκBς activation in keratinocytes and modulates IL-17-IκBς pathway-mediated skin inflammation in an animal model of psoriasis. Currently, the effect of itaconate on regulating macrophage functions and peripheral inflammatory immune responses is well established. However, its effect on microglia (MG) and CNS inflammatory immune responses remains unexplored. Thus, we investigated whether itaconate possesses an immunomodulatory effect on regulating MG activation and CNS inflammation in animal models of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE). Methods: Chronic C57BL/6 EAE was induced followed by DMI treatment. The effect of DMI on disease severity, bloodbrain barrier (BBB) disruption, MG activation, peripheral Th1/Th17 differentiation, and the CNS infiltration of Th1/Th17 cells in EAE was determined. Primary MG was cultured to study the effect of DMI on MG activation. Relapsing-remitting SJL/J EAE was induced to assess the therapeutic effect of DMI. Results: Our results show DMI ameliorated disease severity in the chronic C57BL/6 EAE model. Further analysis of the cellular and molecular mechanisms revealed that DMI mitigated BBB disruption, inhibited MMP3/MMP9 production, suppressed microglia activation, inhibited peripheral Th1/Th17 differentiation, and repressed the CNS infiltration of Th1 and Th17 cells. Strikingly, DMI also exhibited a therapeutic effect on alleviating severity of relapse in the relapsingremitting SJL/J EAE model.

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1. Excitatory postsynaptic currents (EPSCs) following focal afferent stimulation were recorded from patch-clamped magnocellular neurones in a thin-slice preparation of the rat basal forebrain. Evoked EPSCs had a mean decay time constant of 3-81 + 0 09 ms and were reversibly blocked by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 5 AM). and occluded the effect of subsequent application of DA. 6. These results suggest that glutamatergic afferents to magnocellular basal forebrain neurones possess presynaptic D,-like DA receptors, and that activation of these receptors reduces excitatory glutamatergic transmission, probably via an adenylyl cyclase-dependent pathway.

The rate constant for the diffusion of tritiated water across the fetal erythrocyte membrane has been measured as 0.056 -4-SD 0.013 msec. -I The equivalent pore radius calculated by the method of Paganelli and Solomon for the fetal erythrocyte is 3.9 A. The effect of a normal distribution of channel sizes within the erythrocyte membrane is discussed and the theoretical effect of variation of the distribution on the diffusion to bulk flow ratio is evaluated. Since membrane channels of 4 A characterize a variety of membranes, it is suggested that permeability differences may be associated with slight variations in the statistical distribution of the channel sizes within the membrane. The limits of 4-0.5 A standard deviation that qualitative experiments place on the expected variability of channel size will not account for the lower rate of water diffusion in the fetal cell.T h e r e is e x p e r i m e n t a l evidence indicating that the erythrocytes associated with intrauterine a n d n e o n a t a l life possess m e m b r a n e p e r m e a b i l i t y c h a r a cteristics t h a t differ f r o m those found in the adult e r y t h r o c y t e (1). T h e present e x p e r i m e n t s were u n d e r t a k e n to m e a s u r e the rate of diffusion of tritiated w a t e r across the m e m b r a n e of the fetal red blood cell. Using a molecule such as water, which is small relative to the m o l e c u l a r structure of the m e mb r a n e a n d which moves passively across the m e m b r a n e , subtle alterations in the m e m b r a n e structure can be explored a n d p r e s u m a b l y will be reflected in an altered rate of w a t e r diffusion. E X P E R I M E N T A L P R O C E D U R E Materials and MethodsTo determine the kinetic curve for diffusion of tritiated water across the erythrocyte membrane, the method of Paganelli and Solomon (2) was used. In this system blood and tritiated water ( T H O ) labeled buffer are rapidly mixed and forced at a velocity of about 900 cm per sec. along a tube of precisely known diameter. Provision is made for the collection of cell-free samples of the extracellular water at measured distances from the point of mixing. From the velocity of flow of the blood-buffer mixture, the cross-sectional area of the tube, and the distances separating the sampling ports, the duration of T H O diffusion for each sample can be calculated relative to the time 839

A series of structurally constrained analogues based on hybrid compounds containing octahydrobenzo[g or f]quinoline moieties were designed, synthesized and characterized for their binding to dopamine D2 and D3 receptors expressed in HEK-293 cells. Among the newly developed constrained molecules, trans-octahydrobenzo[f]-quinolin-7-ol (8) exhibited the highest affinity for D2 and D3 receptors; the (−)-isomer being the eutomer. Interestingly, this hybrid constrained version 8 showed significant affinity over the corresponding non-hybrid version 1 (representing a constrained version of the aminotetralin structure only) when assayed under same conditions (K i 49.1 and 14.9 nM for 8 vs. 380 and 96.0 nM for 1 at D2 and D3, respectively). Similar results were found with other lead hybrid compounds, indicating a contribution of the piperazine moiety in the observed enhanced affinity. Based on the data of new lead constrained derivatives and other lead hybrid derivatives developed by us, a unique pharmacophore model was proposed consisting of three pharmacophoric centers, two with aromatic/hydrophobic and one with cationic features.