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Background and ObjectivesAlteration of the blood-brain barrier (BBB) at the interface between blood and CNS parenchyma is prominent in most neuroinflammatory diseases. In several neurologic diseases, including cerebral malaria and Susac syndrome, a CD8 T cell–mediated targeting of endothelial cells of the BBB (BBB-ECs) has been implicated in pathogenesis.MethodsIn this study, we used an experimental mouse model to evaluate the ability of a small-molecule perforin inhibitor to prevent neuroinflammation resulting from cytotoxic CD8 T cell–mediated damage of BBB-ECs.ResultsUsing an in vitro coculture system, we first identified perforin as an essential molecule for killing of BBB-ECs by CD8 T cells. We then found that short-term pharmacologic inhibition of perforin commencing after disease onset restored motor function and inhibited the neuropathology. Perforin inhibition resulted in preserved BBB-EC viability, maintenance of the BBB, and reduced CD8 T-cell accumulation in the brain and retina.DiscussionTherefore, perforin-dependent cytotoxicity plays a key role in the death of BBB-ECs inflicted by autoreactive CD8 T cells in a preclinical model and potentially represents a therapeutic target for CD8 T cell–mediated neuroinflammatory diseases, such as cerebral malaria and Susac syndrome.

Recently, the demand for analog and mixed-signal (AMS) integrated circuits (ICs) has increased significantly due to various emerging applications. However, most of the AMS IC layout design efforts are still handled manually at present, which is time-consuming and error-prone. The AMS layout automation level is far from meeting the need for fast layout-circuit performance iterations to accommodate the rapid growth of the market. In general, the methodologies adopted by AMS layout automation tools mainly fall in several different categories: (1) optimization-based approach, which is the most widely used one; (2) template-based approach (e.g., for layout retargeting); and (3) standard cell-based digitalized AMS circuit design and synthesis methodology, which is a new direction preferable in advanced process technology nodes. This dissertation proposes a set of techniques and algorithms to address various practical problems in these major directions of analog and mixed-signal circuit layout automation research. For the direction of the optimization-based AMS IC layout automation, two analog placement algorithms are proposed to improve the layout results from different aspects. Firstly, hierarchical placement techniques for highperformance AMS ICs are proposed, which minimize the critical parasitics in addition to the total area and half-perimeter wirelength, while satisfying the analog placement constraints simultaneously, including symmetry and proximity group constraints. Secondly, an analytical framework is proposed to tackle the device layer-aware analog placement problem, which can effectively reduce the total area and wirelength without degrading the circuit performance, leveraging the fact that it can be beneficial to overlap some devices built by mutually exclusive layers. For the direction of the template-based approach, this dissertation also proposes a new layout retargeting framework. It first applies effective algorithms to extract analog placement constraints from previous quality-approved layouts, including symmetry and regularity constraints. After that, it preserves the prior design expertise during retargeting while exploring different layout topologies to improve the result quality. Furthermore, for both optimization-based and template-based AMS IC layout automation approaches, well island generation persists as a fundamental and unresolved challenge in the post-placement optimization stage. To address the well generation problem, this dissertation proposes a generative adversarial network (GAN) guided framework with a post-refinement stage to mimic the behavior of experienced designers in well generation, leveraging the previous high-quality manually-crafted layouts. Guiding regions for the well islands are first generated by a trained GAN model, after which the results are legalized through post-refinement to satisfy the design rules. Finally, for the standard cell-based digitalized AMS IC design and synthesis methodology, this dissertation presents a scaling compatible, synthesis friendly ring voltage-controlled oscillator (VCO) based ∆Σ analog-to-digital converter (ADC). Its circuit performance improves as process technology advances, and its layout can be fully synthesized by leveraging digital circuit automation tools. It also demonstrates favorable performance compared with the prior digitalized ADCs and in-line performance with state-of-the-art manual designs.

Purpose Tofacitinib is a pan-Janus kinase (JAK) inhibitor that suppresses cytokine signaling and in turn, the cells that participate in inflammatory immunopathogenic processes. We examined the capacity of tofacitinib to inhibit the induction of experimental autoimmune uveitis (EAU) and related immune responses. Methods EAU was induced in B10.A mice with immunization with bovine interphotoreceptor retinoid-binding protein (IRBP), emulsified in complete Freund’s adjuvant (CFA), and a simultaneous injection of pertussis toxin. Tofacitinib, 25 mg/kg, was administered daily, and the vehicle was used for control. EAU development was assessed by histological analysis of the mouse eyes, and related immune responses were assessed by (i) the levels of interferon (IFN)-γ and interleukin (IL)-17, secreted by spleen cells cultured with IRBP; (ii) flow cytometric analysis of intracellular expression by spleen, or eye-infiltrating CD4 or CD8 cells of IFN-γ, IL-17, and their transcription factors, T-bet and RORγt. In addition, the inflammation-related cell markers CD44 and CD62L and Ki67, a proliferation marker, were tested. The proportions of T-regulatory cells expressing FoxP3 were determined by flow cytometric intracellular staining, while levels of antibody to IRBP were measured with enzyme-linked immunosorbent assay (ELISA). Results Treatment with tofacitinib significantly suppressed the development of EAU and reduced the levels of secreted IFN-γ, but not of IL-17. Further, treatment with tofacitinib reduced in the spleen and eye-infiltrating cells the intracellular expression of IFN-γ and its transcription factor T-bet. In contrast, treatment with tofacitinib had essentially no effect on the intracellular expression of IL-17 and its transcription factor, RORγt. The selective effect of tofacitinib treatment was particularly evident in the CD8 population. Treatment with tofacitinib also increased the population of CD44, but reduced the populations of cells producing CD62L and Ki67. Treatment with tofacitinib had no effect on the proportion of FoxP3 producing regulatory cells and on the antibody production to IRBP. Conclusions Treatment with tofacitinib inhibited the development of EAU, reduced the production of IFN-γ, but had essentially no effect on the production of IL-17.

Nanoscopy has now become a real procedure in fluorescence microscopy of living cells. The STED/RESOLFT family of nanoscopy approaches (Hell 2004; Blom 2014) has the best prospects for delivering high speed imaging, but the history of STED includes a continuing struggle to reduce the deactivation power applied, along with difficulties in achieving simultaneous multicolor images. In this manuscript, we present a concept for a similar real-time nanoscopy, using a new class of bipartite probes that separate the luminescent and quenching functions into two coupled molecules. In particular, the STAQ (Superresolution via Transiently Activated Quencher) example we show herein employs the excited state absorbance (not ground state) of the partner to accept energy from and quench the luminescent dye. The result is that much less deactivation power is needed for superresolved (~50nm) imaging. Moreover, the TAQ partner excited by the “donut” beam is shown to quench several different visible dyes via the same mechanism, opening the door to easier multicolor imaging. We demonstrate three dyes sharing the same deactivation and show examples of superresolved multicolor images. We suggest STAQ will facilitate the growth of real-time nanoscopy by reducing confounding photodamage within living cells while expanding the nanoscopist’s palette.