The neuronal ␣7 nicotinic receptor subunit gene (CHRNA7) is partially duplicated in the human genome forming a hybrid gene (CHRFAM7A) with the novel FAM7A gene. The hybrid gene transcript, dup␣7, has been identified in brain, immune cells, and the HL-60 cell line, although its translation and function are still unknown. In this study, dup␣7 cDNA has been cloned and expressed in GH4C1 cells and Xenopus oocytes to study the pattern and functional role of the expressed protein. Our results reveal that dup␣7 transcript was natively translated in HL-60 cells and heterologously expressed in GH4C1 cells and oocytes. Injection of dup␣7 mRNA into oocytes failed to generate functional receptors, but when co-injected with ␣7 mRNA at ␣7/dup␣7 ratios of 5:1, 2:1, 1:1, 1:5, and 1:10, it reduced the nicotine-elicited ␣7 current generated in control oocytes (␣7 alone) by 26, 53, 75, 93, and 94%, respectively. This effect is mainly due to a reduction in the number of functional ␣7 receptors reaching the oocyte membrane, as deduced from ␣-bungarotoxin binding and fluorescent confocal assays. Two additional findings open the possibility that the dominant negative effect of dup␣7 on ␣7 receptor activity observed in vitro could be extrapolated to in vivo situations. (i) Compared with ␣7 mRNA, basal dup␣7 mRNA levels are substantial in human cerebral cortex and higher in macrophages.(ii) dup␣7 mRNA levels in macrophages are down-regulated by IL-1␤, LPS, and nicotine. Thus, dup␣7 could modulate ␣7 receptor-mediated synaptic transmission and cholinergic antiinflammatory response.Neuronal ␣7 nicotinic acetylcholine receptors (␣7 nAChRs) 4 are widely expressed in the central and peripheral nervous systems. In neurons, homomeric ␣7 nAChRs, composed of five ␣7 subunits, modulate neurotransmitter release in presynaptic nerve terminals and induce excitatory impulses in postsynaptic neurons (1-4). Signaling through ␣7 nAChRs in the central nervous system has been associated with neuronal plasticity and cell survival (5-7), although impaired activity of this receptor has been implicated in the pathogenesis of schizophrenia, Alzheimer disease, and depression (8 -12). The presence of ␣7 nAChRs has also been reported in non-neuronal cells such as vascular and brain en-

GABAA receptors are the major inhibitory neurotransmitter receptors in the brain and are the target for many clinically important drugs. Among the many modulatory compounds are also the intravenous anesthetics propofol and etomidate, and barbiturates. The mechanism of receptor modulation by these compounds is of mayor relevance. The site of action of these compounds has been located to subunit interfaces in the intra-membrane region of the receptor. In α1β2γ2 GABAA receptors there are five such interfaces, two β+/α- and one each of α+/β-, α+/γ- and γ+/β- subunit interfaces. We have used reporter mutations located in the second trans-membrane region in different subunits to probe the effects of changes at these subunit interfaces on modulation by propofol, etomidate and pentobarbital. We provide evidence for the fact that each of these compounds either modulates through a different set of subunit interfaces or through the same set of subunit interfaces to a different degree. As a GABAA receptor pentamer harbors two β+/α- subunit interfaces, we used concatenated receptors to dissect the contribution of individual interfaces and show that only one of these interfaces is important for receptor modulation by etomidate.

Nicotine stimulation of α7 nicotinic acetylcholine receptor (α7 nAChR) powerfully inhibits pro-inflammatory cytokine production in lipopolysaccharide (LPS)-stimulated macrophages and in experimental models of endotoxemia. A signaling pathway downstream from the α7 nAChRs, which involves the collaboration of JAK2/STAT3 and NF-κB to interfere with signaling by Toll-like receptors (TLRs), has been implicated in this anti-inflammatory effect of nicotine. Here, we identifiy an alternative mechanism involving interleukin-1 receptor-associated kinase M (IRAK-M), a negative regulator of innate TLR-mediated immune responses. Our data show that nicotine up-regulates IRAK-M expression at the mRNA and protein level in human macrophages, and that this effect is secondary to α7 nAChR activation. By using selective inhibitors of different signaling molecules downstream from the receptor, we provide evidence that activation of STAT3, via either JAK2 and/or PI3K, through a single (JAK2/PI3K/STAT3) or two convergent cascades (JAK2/STAT3 and PI3K/STAT3), is necessary for nicotine-induced IRAK-M expression. Moreover, down-regulation of this expression by small interfering RNAs specific to the IRAK-M gene significantly reverses the anti-inflammatory effect of nicotine on LPS-induced TNF-α production. Interestingly, macrophages pre-exposed to nicotine exhibit higher IRAK-M levels and reduced TNF-α response to an additional LPS challenge, a behavior reminiscent of the ‘endotoxin tolerant’ phenotype identified in monocytes either pre-exposed to LPS or from immunocompromised septic patients. Since nicotine is a major component of tobacco smoke and increased IRAK-M expression has been considered one of the molecular determinants for the induction of the tolerant phenotype, our findings showing IRAK-M overexpression could partially explain the known influence of smoking on the onset and progression of inflammatory and infectious diseases.

This study reveals that the PBMC α7 gene expression level is a clinically relevant marker for CAP activity in sepsis: the higher the α7 expression, the better the inflammation control and the prognosis.

IntroductionCell-free plasma mitochondrial DNA (mt-DNA) and nuclear DNA (n-DNA) are biomarkers with prognostic utility in conditions associated with a high rate of cell death. This exploratory study aimed to determine the plasma levels of both nucleic acids in patients with massive and submassive pulmonary embolism (PE) and to compare them with other biomarkers, such as heart-type fatty acid-binding protein (H-FABP) and troponin I (Tn-I)MethodsThis was a prospective observational study of 37 consecutive patients with massive PE, 37 patients with submassive PE, and 37 healthy subjects. Quantifications of plasma mt-DNA and n-DNA with real-time quantitative polymerase chain reaction (PCR), and plasma H-FABP and Tn-I by commercial assays, were done on blood samples drawn within 4 hours after presentation at the emergency department.ResultsPlasma mt-DNA and n-DNA concentrations were much higher in patients with massive PE (median, 2,970 GE/ml; interquartile range (IQR), 1,050 to 5,485; and 3,325 GE/ml, IQR: 1,080 to 5,790, respectively) than in patients with submassive PE (870 GE/ml and 1,245 GE/ml, respectively; P < 0.01) or controls (185 GE/ml and 520 GE/ml, respectively). Eighteen patients with massive PE died of a PE-related cause by day 15 of observation. Plasma mt-DNA and n-DNA values were 2.3-fold and 1.9-fold higher in the subgroup of nonsurviving patients than in survivors. H-FABP and Tn-I values were also higher in patients with massive PE who died (7.3 ng/ml and 0.023 ng/ml, respectively) than in those who survived (6.4 ng/ml, and 0.016 ng/ml, respectively). By receiver operating curve (ROC) analysis, the best cutoff values for predicting 15-day mortality were 3,380 GE/ml for mt-DNA, 6.8 ng/ml for H-FABP, 3,625 GE/ml for n-DNA, and 0.020 ng/ml for Tn-I, based on the calculated areas under the curve (AUCs) of 0.89 (95% confidence interval (CI), 0.78 to 0.99), 0.76 (95% CI, 0.69 to 093), 0.73 (95% CI, 0.58 to 0.91), and 0.59 (95% CI, 0.41 to 0.79), respectively. By stepwise logistic regression, a plasma mt-DNA concentration greater than 3,380 GE/ml (adjusted odds ratio (OR), 8.22; 95% CI, 1.72 to 39.18; P < 0.001) and a plasma value of H-FBAP >6.8 ng/ml (OR, 5.36; 95% CI, 1.06 to 27.08; P < 0.01) were the only independent predictors of mortality.Conclusionsmt-DNA and H-FBAP might be promising markers for predicting 15-day mortality in massive PE, with mt-DNA having better prognostic accuracy.