Induction and Measurement of Cytotoxic T Lymphocyte Activity.

Cytotoxic T cells (CTLs) are important immune effector cells in the adaptive immune response. It has been well documented that CTLs are important in host immune responses to viral and bacterial intracellular pathogens, tumors, and transplanted tissues. The properties of CTLs have been studied extensively in murine models, and their roles validated in the human setting. Frequently, the presence of these cells correlates well with protective immunity, so the ability to readily measure the activity of these cells is an important immunological measurement. In this unit, several assays are described that are commonly utilized to induce CTLs and to measure CTL activity both in vitro and in vivo. These assays are adaptable to many experimental and/or disease models, and in the case of the in vitro assays can be applied to measure CTL activity in human samples. © 2018 by John Wiley & Sons, Inc.

Polarized localization of voltage-gated Na+ channels is regulated by concerted FGF13 and FGF14 action.

Clustering of voltage-gated sodium channels (VGSCs) within the neuronal axon initial segment (AIS) is critical for efficient action potential initiation. Although initially inserted into both somatodendritic and axonal membranes, VGSCs are concentrated within the axon through mechanisms that include preferential axonal targeting and selective somatodendritic endocytosis. How the endocytic machinery specifically targets somatic VGSCs is unknown. Here, using knockdown strategies, we show that noncanonical FGF13 binds directly to VGSCs in hippocampal neurons to limit their somatodendritic surface expression, although exerting little effect on VGSCs within the AIS. In contrast, homologous FGF14, which is highly concentrated in the proximal axon, binds directly to VGSCs to promote their axonal localization. Single-point mutations in FGF13 or FGF14 abrogating VGSC interaction in vitro cannot support these specific functions in neurons. Thus, our data show how the concerted actions of FGF13 and FGF14 regulate the polarized localization of VGSCs that supports efficient action potential initiation.

CD117, Ki-67, and p53 predict survival in neuroendocrine carcinomas, but not within the subgroup of small cell lung carcinoma.

High-grade neuroendocrine carcinomas (NECs) are aggressive tumors with limited treatment options. Recently, studies have observed that the tyrosine kinase receptor CD117 is often overexpressed in this malignancy. As a result, CD117 has been identified as a target for therapy via the small molecule, tyrosine kinase inhibitor imatinib mesylate. In the present study, 17 low-grade, 4 intermediate-grade, and 76 high-grade NECs were immunostained for CD117, Ki-67, and p53. Overexpression of the three markers was mainly, but not exclusively seen in the high-grade NECs. Patients with overexpression of CD117 and p53 and increased Ki-67 expression showed reduced survival. However, no difference in survival was observed when the same analysis was applied solely to small cell lung cancer patients, the largest subset studied. These findings suggest that overexpression of CD117, p53, and Ki-67 reflects tumor grade and predicts survival in NECs, but fail as prognostic markers in the subset of small cell lung cancer patients.

Reduced cortical BDNF expression and aberrant memory in Carf knock-out mice.

Transcription factors are a key point of convergence between the cell-intrinsic and extracellular signals that guide synaptic development and brain plasticity. Calcium-response factor (CaRF) is a unique transcription factor first identified as a binding protein for a calcium-response element in the gene encoding brain-derived neurotrophic factor (Bdnf). We have now generated Carf knock-out (KO) mice to characterize the function of this factor in vivo. Intriguingly, Carf KO mice have selectively reduced expression of Bdnf exon IV-containing mRNA transcripts and BDNF protein in the cerebral cortex, whereas BDNF levels in the hippocampus and striatum remain unchanged, implicating CaRF as a brain region-selective regulator of BDNF expression. At the cellular level, Carf KO mice show altered expression of GABAergic proteins at striatal synapses, raising the possibility that CaRF may contribute to aspects of inhibitory synapse development. Carf KO mice show normal spatial learning in the Morris water maze and normal context-dependent fear conditioning. However they have an enhanced ability to find a new platform location on the first day of reversal training in the water maze and they extinguish conditioned fear more slowly than their wild-type littermates. Finally, Carf KO mice show normal short-term (STM) and long-term memory (LTM) in a novel object recognition task, but exhibit impairments during the remote memory phase of testing. Together, these data reveal novel roles for CaRF in the organization and/or function of neural circuits that underlie essential aspects of learning and memory.