Acetylcholine (ACh) regulates vital functions of T cells by acting on the nicotinic and muscarinic classes of cholinergic receptors, nAChR and mAChRs, respectively. This study was performed in murine splenic T cells. In freshly isolated CD4 and CD8 T cells, we detected mRNAs encoding a5, a9, a10, b1, b2, b4 nAChR subunits and M 1 , M 3 , M 4 and M 5 mAChR subtypes, whereas a2 was detected only in CD8 T cells. In vitro activation of CD4 T cells through T-cell receptor (TCR)/CD3 cross-linking was associated with the appearance of a4 and a7, upregulation of a5, a10, b4, M 1 and M 5 and downregulation of a9 and b2, whereas in vitro activation of CD8 T cells also featured the appearance of a4 and a7, as well as upregulation of a2, a5, b4, M 1 and M 4 , and downregulation of a10, b1, b2 and M 3 . In vitro polarization toward T helper (Th) 1 lineage was associated with a decrease of b2, b4 and M 3 expression; that toward Th2 cells with downregulation of a9 and M 3 , and upregulation of M 1 and M 5 ; and that toward Th17 phenotype with downregulation of a9, a10, b2 and M 3 mAChR. Polarized T cells also expressed a4, but not a1, a2, a3, a6, b3 or M 2 . To determine the role of cholinergic receptors in mediating the immunoregulatory action of autocrine/paracrine ACh, we analyzed the effects of nicotinic and muscarinic agonists ± antagonists on cytokine production in the CD4 þ CD62L þ T cells co-stimulated via TCR/CD3 cross-linking. The nicotinergic stimulation upregulated interferon-g (IFN-g) and downregulated interleukin (IL)-17 secretion, whereas the muscarinic stimulation enhanced IL-10 and IL-17 and inhibited INF-g secretion. These results demonstrated plasticity of the T-cell cholinergic system.

Ulcerative colitis (UC) and Crohn’s disease (CD) are two forms of chronic inflammatory bowel disease. CD4 T cells play a central role in the pathogenesis of both diseases. Smoking affects both UC and CD but with opposite effects, ameliorating UC and worsening CD. We hypothesized that the severity of gut inflammation could be modulated through T-cell nicotinic acetylcholine receptors (nAChRs) and that the exact clinical outcome would depend on the repertoire of nAChRs on CD4 T cells mediating each form of colitis. We measured clinical and immunologic outcomes of treating BALB/c mice with oxazolone- and TNBS-induced colitides by nicotine. Nicotine attenuated oxazolone colitis, which was associated with increased percentage of colonic Tregs and a reduction of Th17 cells. TCR stimulation of naïve CD4+CD62L+ T cells in the presence of nicotine upregulated expression of Foxp3. In marked contrast, nicotine worsened TNBS colitis, and this was associated with increased Th17 cells among colonic CD4 T cells. Nicotine upregulated IL-10 and inhibited IL-17 production, which could be abolished by exogenous IL-12 that also abolished the nicotine-dependent upregulation of Tregs. The dichotomous action of nicotine resulted from the up- and downregulation of anti-inflammatory α7 nAChR on colonic CD4 T cells induced by cytokines characteristic of the inflammatory milieu in oxazolone (IL-4), and TNBS (IL-12) colitis, respectively. These findings help explain the dichotomous effect of smoking in patients with UC and CD, and underscore the potential for nicotinergic drugs in regulating colonic inflammation.

SLURP-1 (secreted mammalian Ly-6/urokinase plasminogen activator receptor-related protein-1) is a novel auto/paracrine cholinergic peptide that can bind to α(7)-nicotinic acetylcholine receptor (nAChR), a high Ca(2+)-permeable ion channel coupled to regulation of nuclear factor-κB (NF-κB) expression. Elucidation of intracellular signaling events elicited by SLURP-1 is crucial for understanding the molecular mechanism of functioning of this novel hormone-like peptide that alters vital cell functions and can protect from tumorigenic transformation. In this study, we sought to dissect out the role of α(7)-nAChR in mediating the biologic effects of recombinant SLURP-1 on the immortalized line of human oral keratinocytes Het-1A. A multifold upregulation of the NF-κB expression at the mRNA and protein levels by SLURP-1 was only slightly diminished due to elimination of Na(+), whereas in Ca(2+)-free medium the effect of SLURP-1 was inhibited by >50%. Both in the absence of extracellular Ca(2+) and in the presence of Cd(2+) or Zn(2+), the SLURP-1-dependent elevation of NF-κB was almost completely blocked by inhibiting MEK1 activity. Downstream of α(7)-nAChR, the SLURP-1 signaling coupled to upregulation of NF-κB also involved Jak2 as well as Ca(2+)/calmodulin-dependent kinase II (CaMKII) and protein kinase C (PKC), whose inhibition significantly (P < 0.05) reduced the SLURP-1-induced upregulation of NF-κB. The obtained results indicated that activation of α(7)-nAChR by SLURP-1 leads to upregulation of the NF-κB gene expression due to activation of the Raf-1/MEK1/ERK1/2 cascade that proceeds via two complementary signaling pathways. One is mediated by the Ca(2+)-entry dependent CaMKII/PKC activation and another one by Ca(2+)-independent involvement of Jak2. Thus, there exists a previously not appreciated network of noncanonical auto/paracrine ligands of nAChR of the Ly-6 protein family, which merits further investigations.

The search for genetic variants associated with ovarian cancer risk has focused on pathways including sex steroid hormones, DNA repair, and cell cycle control. The Ovarian Cancer Association Consortium (OCAC) identified 10 single-nucleotide polymorphisms (SNPs) in genes in these pathways, which had been genotyped by Consortium members and a pooled analysis of these data was conducted. Three of the 10 SNPs showed evidence of an association with ovarian cancer at Pp0.10 in a log-additive model: rs2740574 in CYP3A4 (P ¼ 0.011), rs1805386 in LIG4 (P ¼ 0.007), and rs3218536 in XRCC2 (P ¼ 0.095). Additional genotyping in other OCAC studies was undertaken and only the variant in CYP3A4, rs2740574, continued to show an association in the replication data among homozygous carriers: OR homozygous(hom) ¼ 2.50 (95% CI 0.54-11.57, P ¼ 0.24) with 1406 cases and 2827 controls. Overall, in the combined data the odds ratio was 2.81 among carriers of two copies of the minor allele (95% CI 1.20 -6.56, P ¼ 0.017, p het across studies ¼ 0.42) with 1969 cases and 3491 controls. There was no association among heterozygous carriers. CYP3A4 encodes a key enzyme in oestrogen metabolism and our finding between rs2740574 and risk of ovarian cancer suggests that this pathway may be involved in ovarian carcinogenesis. Additional follow-up is warranted.

We describe and explore the capability of a photothermal (PT) assay with modified schematics for highly sensitive detection of individual cell response to antitumor drug impact in vitro. Specifically, we used the nonlinear differential PT test to measure distinctive changes of specific PT parameters after exposure of KB3 carcinoma cells to the antitumor drug vinblastine in the broad concentration range of 10(-10) to 300 nM. Verification of the PT assay was performed by comparison with multidrug-resistant cells and comparison with conventional assays evaluating cell viability, cytochrome c release, apoptosis induction, and cell size. We demonstrate that this system is capable of detecting drug-induced signals at a concentration threshold sensitivity at least seven orders of magnitude better than existing assays. We anticipate that this technique may serve as a convenient and rapid analytical tool to evaluate the presence of intracellular drug, with applications in high throughput screening assays and for studying drug uptake and distribution in more complex biological or clinical samples.