Several genes coding for different cytokines may affect host susceptibility to tuberculosis. This study investigates the relationship of the single base change polymorphic variants identified in the first intron of interferon-gamma (+874 T/A) and in the promoter region of interleukin-10 gene (-1,082 G/A), with cytokine production by peripheral blood mononuclear cells and tuberculosis susceptibility. We studied a Spanish population of 113 patients with culture-proven pulmonary tuberculosis, 207 healthy close contacts (125 tuberculin reactive and 82 tuberculin negative), and 100 healthy tuberculin-negative control subjects. Multiple logistic regression analysis showed that individuals homozygous for the interferon-gamma (+874) A allele had a 3.75-fold increased risk of developing tuberculosis (95% confidence interval, 2.26-6.23, p = 0.0017). Stimulated production of interferon-gamma by peripheral mononuclear cells from patients with genotype AA was depressed compared with that of non-AA homozygotes at the time of diagnosis and after completion of therapy. Multivariate analysis showed that the presence of an AA genotype and the absolute number of lymphocytes were the only independent predictors of interferon-gamma production. In contrast, the different rates of interleukin-10 production associated with the interleukin-10 polymorphism did not affect susceptibility to tuberculosis. Thus, a genetic defect in the production of interferon-gamma in individuals homozygous for the (+874) A allele could contribute to their increased risk of developing tuberculosis.

The relationship between fluctuating cytokine concentrations in plasma and the outcome of sepsis is complex. We postulated that early measurement of the activation of nuclear factor B (NF-B), a transcriptional regulatory protein involved in proinflammatory cytokine expression, may help to predict the outcome of sepsis. We determined NF-B activation in peripheral blood mononuclear cells of 34 patients with severe sepsis (23 survivors and 11 nonsurvivors) and serial concentrations of inflammatory cytokines (interleukin-6, interleukin-1, and tumor necrosis factor) and various endogenous antagonists in plasma. NF-B activity was significantly higher in nonsurvivors and correlated strongly with the severity of illness (APACHE II score), although neither was related to the cytokine levels. Apart from NF-B activity, the interleukin-1 receptor antagonist was the only cytokine tested whose level in plasma was of value in predicting mortality by logistic regression analysis. These results underscore the prognostic value of early measurement of NF-B activity in patients with severe sepsis.Many reports have focused on aspects of the proinflammatory cytokine network, which is believed to be central to the pathophysiology of the sepsis syndrome (5,8). However, the cytokine responses in patients with sepsis appears to vary so much between individuals (10) that the prognostic usefulness of circulating cytokine concentrations is often less than that of clinical variables, such as the acute physiology and chronic health evaluation (APACHE) II or III (9). Other studies indicate that the problem in overwhelming sepsis is not that inflammatory cytokines are expressed but, rather, that their expression is not properly modulated by anti-inflammatory mediators (16,17). Recent investigations by others (3) and ourselves (1) searching for new clinically reliable markers in patients with sepsis have shown that circulating leptin levels, whose secretion is closely linked to the activation of the cytokine cascade (1), may help to predict mortality in sepsis and septic shock.Among several transcriptional regulatory factors involved in immunoregulatory genes expression, nuclear factor kappa B (NF-B) acts at a critical step for directing the transcription of many proinflammatory genes in animal models of inflammatory diseases (6, 7). Investigations regarding the role of NF-B in human inflammatory diseases are scarce (2, 15). So far, no study has aimed to examine in patients with sepsis the relationship between the concentrations of some components of the proinflammatory and anti-inflammatory cytokine response in plasma, NF-B expression in peripheral blood mononuclear cells, and clinical outcome. We hypothesized that severe, fatal sepsis could be distinguished from less severe sepsis by demonstrating greater NF-B activation and decreased anti-inflammatory response. Thus, this study compared the prognostic value of combining measurements of NF-B activity in circulating blood cells and the cytokine profile in plasma in patients with severe sepsis. ...

Leptin production is increased in rodents by administration of endotoxin or cytokines. To investigate whether circulating leptin is related to cytokine release and survival in human sepsis, plasma concentrations of leptin, interleukin (IL)-6, IL-1beta, tumor necrosis factor (TNF)-alpha, soluble TNF receptor type I, IL-1 receptor antagonist (IL-1ra), and the inflammatory modulator IL-10 were measured as soon as severe sepsis (n=28) or septic shock (n=14) developed and every 6 h for 24 h. Patients with sepsis or septic shock had leptin concentrations 2.3- and 4.2-fold greater, respectively, than the control group. There was an independent association for leptin with IL-1ra and IL-10 in both patient groups. By discriminant analysis, leptin and IL-6 were independent predictors of death. These findings suggest that increases in leptin levels may be a host defense mechanism during sepsis.

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-

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Sepsis-associated encephalopathy (SAE) is a frequent but poorly understood neurological complication in sepsis that negatively influences survival. Here we present clinical and experimental evidence that this brain dysfunction may be related to altered neurotransmission produced by inflammatory mediators. Compared with septic patients, SAE patients had higher interleukin-1␤ (IL-1␤) plasma levels; interestingly, these levels decreased once the encephalopathy was resolved. A putative IL-1␤ effect on type A ␥-aminobutyric acid receptors (GABA A Rs), which mediate fast synaptic transmission in most cerebral inhibitory synapses in mammals, was investigated in cultured hippocampal neurons and in Xenopus oocytes expressing native or foreign rat brain GABA A Rs, respectively. Confocal images in both cell types revealed that IL-1␤ increases recruitment of GABA A Rs to the cell surface. Moreover, brief applications of IL-1␤ to voltage-clamped oocytes yielded a delayed potentiation of the GABA-elicited chloride currents (I GABA ); this effect was suppressed by IL-1ra, the natural IL-1 receptor (IL-1RI) antagonist. Western blot analysis combined with I GABA recording and confocal images of GABA A Rs in oocytes showed that IL-1␤ stimulates the IL-1RI-dependent phosphatidylinositol 3-kinase activation and the consequent facilitation of phospho-Akt-mediated insertion of GABA A Rs into the cell surface. The interruption of this signaling pathway by specific phosphatidylinositol 3-kinase or Akt inhibitors suppresses the cytokine-mediated effects on GABA A R, whereas activation of the conditionally active form of Akt1 (myr-Akt1.ER*) with 4-hydroxytamoxifen reproduces the effects. These findings point to a previously unrecognized signaling pathway that connects IL-1␤ with increased "GABAergic tone." We propose that through this mechanism IL-1␤ might alter synaptic strength at central GABAergic synapses and so contribute to the cognitive dysfunction observed in SAE.

SUMMARY This study aims to determine the influence of the polymorphism within the intron 2 of the interleukin‐1 receptor antagonist gene (IL‐1RN*) on the outcome of severe sepsis, and to assess its functional significance by correlating this polymorphism with the total production of interleukin‐1 receptor antagonist (IL‐1Ra) protein determined in stimulated peripheral blood mononuclear cells (PBMC). A group of 78 patients with severe sepsis (51 survivors and 27 nonsurvivors) was compared with a healthy control group of 130 blood donors, and 56 patients with uncomplicated pneumonia. We found a significant association between IL‐1RN* polymorphism and survival. Thus, after adjusting for age and APACHE II score, multiple logistic regression analysis showed that patients homozygotes for the allele *2 had a 6·47‐fold increased risk of death (95% CI 1·01–41·47, P = 0·04). Besides, compared with patients homozygous or heterozygous for the allele *1, IL‐1RN*2 homozygotes produced significantly lower levels of IL‐1Ra from their PBMC. Our results suggest that insufficient production of this cytokine might contribute, among other factors, to the higher mortality rate found in severe sepsis patients with the IL‐1RN*2 homozygous genotype.

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.