Tau has become an attractive alternative target for passive immunotherapy efforts for Alzheimer’s disease (AD). The anatomical distribution and extent of tau pathology correlate with disease course and severity better than other disease markers to date. We describe here the generation, preclinical characterization, and phase 1 clinical characterization of semorinemab, a humanized anti-tau monoclonal antibody with an immunoglobulin G4 (igG4) isotype backbone. Semorinemab binds all six human tau isoforms and protects neurons against tau oligomer neurotoxicity in cocultures of neurons and microglia. In addition, when administered intraperitoneally once weekly for 13 weeks, murine versions of semorinemab reduced the accumulation of tau pathology in a transgenic mouse model of tauopathy, independent of antibody effector function status. Semorinemab also showed clear evidence of target engagement in vivo, with increases in systemic tau concentrations observed in tau transgenic mice, nonhuman primates, and humans. Higher concentrations of systemic tau were observed after dosing in AD participants compared to healthy control participants. No concerning safety signals were observed in the phase 1 clinical trial at single doses up to 16,800 mg and multiple doses totaling 33,600 mg in a month.
Cannabinoids exhibit broad immune modulating activity by targeting many cell types within the immune system, including T cells, which exhibit sensitivity, as evidenced by altered activation, proliferation, and cytokine expression. As a result of the critical role calcium plays in T cell function coupled with previous findings demonstrating disruption of the calcium-regulated transcription factor, nuclear factor of activated T cells, by cannabinoid treatment, the objective of the present investigation was to perform an initial characterization of the role of the cannabinoid receptors in the regulation of the intracellular calcium concentration ([Ca(2+)](i)) by delta(9)-tetrahydrocannabinol (delta(9)-THC) in T lymphocytes. Here, we demonstrate that delta(9)-THC robustly elevates [Ca(2+)](i) in purified murine splenic T cells and in the human peripheral blood acute lymphoid leukemia (HPB-ALL) human T cell line but only minimally elevates [Ca(2+)](i) in Jurkat E6-1 (dysfunctional cannabinoid receptor 2-expressing) human T cells. Removal of extracellular calcium severely attenuated the delta(9)-THC-mediated rise in [Ca(2+)](i) in murine splenic T cells and HPB-ALL cells. Pretreatment with cannabinoid receptor antagonists, SR144528 and/or SR141716A, led to an attenuation of delta(9)-THC-mediated elevation in [Ca(2+)](i) in splenic T cells and HPB-ALL cells but not in Jurkat E6-1 cells. Furthermore, pretreatment of HPB-ALL cells with SR144528 antagonized the small rise in [Ca(2+)](i) elicited by delta(9)-THC in the absence of extracellular calcium. These findings suggest that delta(9)-THC induces an influx of extracellular calcium in resting T cells in a cannabinoid receptor-dependent manner.
It has been demonstrated previously that cannabinol (CBN) differentially modulates interleukin-2 (IL-2) protein secretion by T cells with a corresponding change in extracellular signalregulated kinase activity. The objective of the present studies was to further investigate the molecular mechanism by which CBN enhances IL-2 gene expression using the EL4 T cell line. We demonstrate here that steady-state IL-2 mRNA expression was significantly enhanced by CBN in a concentration-dependent manner in EL4 cells activated with suboptimal concentrations of phorbol-12-myristate-13-acetate (2-10 nM). Concordantly, a marked increase was observed in nuclear factor of activated T cells (NF-AT) DNA binding activity to the IL-2 distal NF-AT site, but not to nuclear factor for immunoglobulin chain in B cells or activator protein 1 motifs. Transient transfection of EL4 cells with a reporter gene under the control of multiple IL-2 distal NF-AT motifs exhibited increased transcriptional activity by CBN in suboptimally activated cells. In addition, the CBNmediated enhancement of IL-2 protein secretion and the transcriptional activity of the IL-2 distal NF-AT reporter gene was abrogated by the calcium/calmodulin-dependent protein kinase inhibitor KN93, but not by the CB2 receptor antagonist SR144528. Enhancement of IL-2 was also demonstrated with CP55940, ⌬ 9 -tetrahydrocannabinol, and cannabidiol, thus suggesting that the phenomenon is not unique to CBN. Collectively, these results suggest that increased IL-2 secretion by CBN is mediated through the enhancement of IL-2 gene transcription by activation of NF-AT in a CB1/CB2-independent manner.
We have reported previously that Delta9-tetrahydrocannabinol (Delta9-THC) treatment of resting human and murine splenic T cells robustly elevated intracellular calcium ([Ca2+]i). The objective of the present investigation was to examine the putative role of [Ca2+]i store depletion and store-operated calcium (SOC) and receptor-operated cation (ROC) channels in the mechanism by which Delta9-THC increases [Ca2+]i in the cannabinoid-2 receptor-expressing human peripheral blood-acute lymphoid leukemia (HPB-ALL) human T cell line. By using the smooth endoplasmic reticulum Ca2+-ATPase pump inhibitor, thapsigargin, and the ryanodine receptor antagonist, 8-bromo-cyclic adenosine diphosphate ribose, we demonstrate that the Delta9-THC-mediated elevation in [Ca2+]i occurs independently of [Ca2+]i store depletion. Furthermore, the ROC channel inhibitor, SK&F 96365 was more efficacious at attenuating the Delta9-THC-mediated elevation in [Ca2+]i than SOC channel inhibitors, 2-aminoethoxydiphenyl borate and La3+. Recently, several members of the transient receptor potential canonical (TRPC) channel subfamily have been suggested to operate as SOC or ROC channels. In the present studies, treatment of HPB-ALL cells with 1-oleoyl-2-acetyl-sn-glycerol (OAG), a cell-permeant analog of diacylglycerol (DAG), which gates several members of the TRPC channel subfamily, rapidly elevated [Ca2+]i, as well as prevented a subsequent, additive elevation in [Ca2+]i by Delta9-THC, independent of protein kinase C. Reverse transcriptase-polymerase chain reaction analysis for TRPC1-7 showed that HPB-ALL cells express detectable mRNA levels of only TRPC1. Finally, small interference RNA knockdown of TRPC1 attenuated the Delta9-THC-mediated elevation of [Ca2+]i. Collectively, these results suggest that Delta9-THC-induced elevation in [Ca2+]i is attributable entirely to extracellular calcium influx, which is independent of [Ca2+]i store depletion, and is mediated, at least partially, through the DAG-sensitive TRPC1 channels.
BackgroundEngagement of the β2 integrin, lymphocyte function-associated antigen-1 (LFA-1), results in stabilization of T cell mRNA transcripts containing AU-rich elements (AREs) by inducing rapid nuclear-to-cytosolic translocation of the RNA-stabilizing protein, HuR. However, little is known regarding integrin-induced signaling cascades that affect mRNA catabolism. This study examines the role of the GTPases, Rac 1 and Rac 2, and their downstream effectors, in the LFA-1-induced effects on mRNA.Methodology/Principal FindingsEngagement of LFA-1 to its ligand, ICAM-1, in human peripheral T cells resulted in rapid activation of Rac1 and Rac2. siRNA-mediated knockdown of either Rac1 or Rac2 prevented LFA-1-stimulated stabilization of the labile transcripts encoding IFN-γ and TNF-α, and integrin mediated IFN-γ mRNA stabilization was absent in T cells obtained from Rac2 gene-deleted mice. LFA-1 engagement-induced translocation of HuR and stabilization of TNF- α mRNA was lost in Jurkat cells deficient in the Rac guanine nucleotide exchange factor Vav-1 (J.Vav1). The transfection of J.Vav1 cells with constitutively active Rac1 or Rac2 stabilized a labile β-globin reporter mRNA, in a HuR-dependent manner. Furthermore, LFA-1-mediated mRNA stabilization and HuR translocation in mouse splenic T cells was dependent on the phosphorylation of the mitogen-activated protein kinase kinase, MKK3, and its target MAP kinase p38MAPK, and lost in T cells obtained from MKK3 gene-deleted mice.Conclusions/SignificanceCollectively, these results demonstrate that LFA-1-induced stabilization of ARE-containing mRNAs in T cells is dependent on HuR, and occurs through the Vav-1, Rac1/2, MKK3 and p38MAPK signaling cascade. This pathway constitutes a molecular switch that enhances immune and pro-inflammatory gene expression in T cells undergoing adhesion at sites of activation and effector function.
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