Temporal lobe epilepsy (TLE) is accompanied by an abnormal location of granule cells in the dentate gyrus. Using a rat model of complex febrile seizures, which are thought to be a precipitating insult of TLE later in life, we report that aberrant migration of neonatal-generated granule cells results in granule cell ectopia that persists into adulthood. Febrile seizures induced an upregulation of GABA(A) receptors (GABA(A)-Rs) in neonatally generated granule cells, and hyperactivation of excitatory GABA(A)-Rs caused a reversal in the direction of granule cell migration. This abnormal migration was prevented by RNAi-mediated knockdown of the Na(+)K(+)2Cl(-) co-transporter (NKCC1), which regulates the excitatory action of GABA. NKCC1 inhibition with bumetanide after febrile seizures rescued the granule cell ectopia, susceptibility to limbic seizures and development of epilepsy. Thus, this work identifies a previously unknown pathogenic role of excitatory GABA(A)-R signaling and highlights NKCC1 as a potential therapeutic target for preventing granule cell ectopia and the development of epilepsy after febrile seizures.

BackgroundHigh C reactive protein (CRP) levels have been reported to be associated with a poor clinical outcome in a number of malignancies and with programmed cell death protein 1 immune checkpoint blockade in patients with advanced cancer. Little is known about the direct effects of CRP on adaptive immunity in cancer. Therefore, we investigated how CRP impacted the function of T cells and dendritic cells (DCs) from patients with melanoma.MethodsThe effects of CRP on proliferation, function, gene expression and phenotype of patient T cells and DCs, and expansion of MART-1 antigen-specific T cells were analyzed by multicolor flow cytometry and RNA-seq. Additionally, serum CRP levels at baseline from patients with metastatic melanoma treated on the Checkmate-064 clinical trial were assessed by a Luminex assay.ResultsIn vitro, CRP inhibited proliferation, activation-associated phenotypes and the effector function of activated CD4+ and CD8+ T cells from patients with melanoma. CRP-treated T cells expressed high levels of interleukin-1β, which is known to enhance CRP production from the liver. CRP also suppressed formation of the immune synapse and inhibited early events in T-cell receptor engagement. In addition, CRP downregulated the expression of costimulatory molecules on mature DCs and suppressed expansion of MART-1-specific CD8+ T cells in a dose-dependent manner by impacting on both T cells and antigen-presenting cells. High-serum CRP levels at baseline were significantly associated with a shorter survival in both nivolumab-treated and ipilimumab-treated patients.ConclusionsThese findings suggest that high levels of CRP induce an immunosuppressivemilieuin melanoma and support the blockade of CRP as a therapeutic strategy to enhance immune checkpoint therapies in cancer.Trial registration numberNCT01783938andNCT02983006.

Abstract. Low-cost, simple procedures for organotypic tissue cultures are desirable for highthroughput biological experiments such as large-scale medical / drug screening. We present a practical and economical method to cultivate brain slices using hydrophilic filtration membranes. With a cost reduction of more than 90%, this technique allows us to prepare hippocampal slice cultures that are morphologically and functionally indistinguishable from those obtained by the widely used Millicell-CM ® method.

Proper axonal targeting is fundamental to the establishment of functional neural circuits. The hippocampal mossy fibres normally project towards the CA3 region. In the hippocampi of patients with temporal lobe epilepsy and related animal models, however, mossy fibres project towards the molecular layer and produce the hyperexcitable recurrent networks. The cellular and molecular mechanisms underlying this aberrant axonal targeting, known as mossy fibre sprouting, remain unclear. Netrin-1 attracts or repels axons depending on the composition of its attraction-mediating receptor, deleted in colorectal cancer, and its repulsion-mediating receptor, uncoordinated-5, on the growth cone; but the roles of netrin-1-dependent guidance in pathological conditions are largely unknown. In this study, we examined the role of netrin-1 and its receptors in mossy fibre guidance and report that enhanced neuronal activity changes netrin-1-mediated cell targeting by the axons under hyperexcitable conditions. Netrin-1 antibody or Dcc ribonucleic acid interference attenuated mossy fibre growth towards CA3 in slice overlay assays. The axons were repelled from CA3 and ultimately innervated the molecular layer when hyperactivity was pharmacologically introduced. We first hypothesized that a reduction in netrin-1 expression in CA3 underlies the phenomenon, but found that its expression was increased. We then examined two possible activity-dependent changes in netrin-1 receptor expression: a reduction in the deleted in colorectal cancer receptor and induction of uncoordinated-5 receptor. Hyperactivity did not affect the surface expression of the deleted in colorectal cancer receptor on the growth cone, but it increased that of uncoordinated-5A, which was suppressed by blocking cyclic adenosine monophosphate signalling. In addition, Dcc knockdown did not affect hyperactivity-induced mossy fibre sprouting in the slice cultures, whereas Unc5a knockdown rescued the mistargeting. Thus, netrin-1 appears to attract mossy fibres via the deleted in colorectal cancer receptor, while it repels them via cyclic adenosine monophosphate-induced uncoordinated-5A under hyperexcitable conditions, resulting in mossy fibre sprouting.

Overexpression of EPHA2 has been observed in multiple cancers and reported to be associated with poor prognosis. Here, we produced an afucosylated humanized anti-EPHA2 monoclonal antibody (mAb), DS-8895a for cancer treatment. The antibody recognizes the extracellular juxtamembrane region of EPHA2 and therefore can bind to both full-length and truncated forms of EPHA2, which are anchored to cell membranes and recently reported to be produced by post-translational cleavage in tumors. DS-8895a exhibited markedly increased antibody dependent cellular cytotoxicity (ADCC) in vitro and also inhibited tumor growth in EPHA2-positive human breast cancer MDA-MB-231 and human gastric cancer SNU-16 xenograft mouse models. Moreover, DS-8895a in combination with cisplatin (CDDP) showed better efficacy than each of the monotherapies did in the human gastric cancer model. These results suggest that a novel antibody, DS-8895a has therapeutic potential against EPHA2-expressing tumors.