Alpha-7 Nicotinic Receptor Agonist Protects Mice Against Pulmonary Emphysema Induced by Elastase.

Pulmonary emphysema is a primary component of chronic obstructive pulmonary disease (COPD), a life-threatening disorder characterized by lung inflammation and restricted airflow, primarily resulting from the destruction of small airways and alveolar walls. Cumulative evidence suggests that nicotinic receptors, especially the α7 subtype (α7nAChR), is required for anti-inflammatory cholinergic responses. We postulated that the stimulation of α7nAChR could offer therapeutic benefits in the context of pulmonary emphysema. To investigate this, we assessed the potential protective effects of PNU-282987, a selective α7nAChR agonist, using an experimental emphysema model. Male mice (C57BL/6) were submitted to a nasal instillation of porcine pancreatic elastase (PPE) (50 µl, 0.667 IU) to induce emphysema. Treatment with PNU-282987 (2.0 mg/kg, ip) was performed pre and post-emphysema induction by measuring anti-inflammatory effects (inflammatory cells, cytokines) as well as anti-remodeling and anti-oxidant effects. Elastase-induced emphysema led to an increase in the number of α7nAChR-positive cells in the lungs. Notably, both groups treated with PNU-282987 (prior to and following emphysema induction) exhibited a significant decrease in the number of α7nAChR-positive cells. Furthermore, both groups treated with PNU-282987 demonstrated decreased levels of macrophages, IL-6, IL-1ß, collagen, and elastic fiber deposition. Additionally, both groups exhibited reduced STAT3 phosphorylation and lower levels of SOCS3. Of particular note, in the post-treated group, PNU-282987 successfully attenuated alveolar enlargement, decreased IL-17 and TNF-α levels, and reduced the recruitment of polymorphonuclear cells to the lung parenchyma. Significantly, it is worth noting that MLA, an antagonist of α7nAChR, counteracted the protective effects of PNU-282987 in relation to certain crucial inflammatory parameters. In summary, these findings unequivocally demonstrate the protective abilities of α7nAChR against elastase-induced emphysema, strongly supporting α7nAChR as a pivotal therapeutic target for ameliorating pulmonary emphysema.

Aerobic exercise training engages cholinergic signaling to improve emphysema induced by cigarette smoke exposure in mice.

Lung inflammation is modulated by cholinergic signaling and exercise training protects mice against pulmonary emphysema development; however, whether exercise training engages cholinergic signaling is unknown. AIMS: As cholinergic signaling is directly linked to the vesicular acetylcholine transporter (VAChT) levels, we evaluated whether the effects of aerobic exercise training depend on the VAChT levels in mice with pulmonary emphysema. MAIN METHODS: Wild-type (WT) and mutant (KDHOM) mice (65-70% of reduction in VAChT levels) were exposed to cigarette smoke (30 min, 2×/day, 5×/week, 12 weeks) and submitted or not to aerobic exercise training on a treadmill (60 min/day, 5×/week, 12 weeks). Lung function and inflammation were evaluated. KEY FINDINGS: Cigarette smoke reduced body mass in mice (p < 0.001) and increased alveolar diameter (p < 0.001), inflammation (p < 0.001) and collagen deposition (p < 0.01) in lung tissue. Both trained groups improved their performance in the final physical test compared to the initial test (p < 0.001). In WT mice, exercise training protected against emphysema development (p < 0.05), reduced mononuclear cells infiltrate (p < 0.001) and increased MAC-2 positive cells in lung parenchyma (p < 0.05); however, these effects were not observed in KDHOM mice. The exercise training reduced iNOS-positive cells (p < 0.001) and collagen fibers deposition (p < 0.05) in lung parenchyma of WT and KDHOM mice, although KDHOM mice showed higher levels of iNOS-positive cells. SIGNIFICANCE: Our data suggest that the protective effects of aerobic exercise training on pulmonary emphysema are, at least in part, dependent on the integrity of the lung cholinergic signaling.

Neuronal cholinergic signaling constrains norepinephrine activity in the heart.

It is well known that cholinergic hypofunction contributes to cardiac pathology, yet, the mechanisms involved remain unclear. Our previous study has shown that genetically engineered model of cholinergic deficit, the vesicular acetylcholine transporter knockdown homozygous (VAChT KDHOM) mice, exhibit pathological cardiac remodeling and a gradual increase in cardiac mass with aging. Given that an increase in cardiac mass is often caused by adrenergic hyperactivity, we hypothesized that VAChT KDHOM mice might have an increase in cardiac norepinephrine (NE) levels. We thus investigated the temporal changes in NE content in the heart from 3-, 6-, and 12-mo-old VAChT mutants. Interestingly, mice with cholinergic hypofunction showed a gradual elevation in cardiac NE content, which was already increased at 6 mo of age. Consistent with this finding, 6-mo-old VAChT KDHOM mice showed enhanced sympathetic activity and a greater abundance of tyrosine hydroxylase positive sympathetic nerves in the heart. VAChT mutants exhibited an increase in peak calcium transient, and mitochondrial oxidative stress in cardiomyocytes along with enhanced G protein-coupled receptor kinase 5 (GRK5) and nuclear factor of activated T-cells (NFAT) staining in the heart. These are known targets of adrenergic signaling in the cell. Moreover, vagotomized-mice displayed an increase in cardiac NE content confirming the data obtained in VAChT KDHOM mice. Establishing a causal relationship between acetylcholine and NE, VAChT KDHOM mice treated with pyridostigmine, a cholinesterase inhibitor, showed reduced cardiac NE content, rescuing the phenotype. Our findings unveil a yet unrecognized role of cholinergic signaling as a modulator of cardiac NE, providing novel insights into the mechanisms that drive autonomic imbalance.

Long-term endogenous acetylcholine deficiency potentiates pulmonary inflammation in a murine model of elastase-induced emphysema.

Acetylcholine (ACh), the neurotransmitter of the cholinergic system, regulates inflammation in several diseases including pulmonary diseases. ACh is also involved in a non-neuronal mechanism that modulates the innate immune response. Because inflammation and release of pro-inflammatory cytokines are involved in pulmonary emphysema, we hypothesized that vesicular acetylcholine transport protein (VAChT) deficiency, which leads to reduction in ACh release, can modulate lung inflammation in an experimental model of emphysema. Mice with genetical reduced expression of VAChT (VAChT KDHOM 70%) and wild-type mice (WT) received nasal instillation of 50 uL of porcine pancreatic elastase (PPE) or saline on day 0. Twenty-eight days after, animals were evaluated. Elastase instilled VAChT KDHOM mice presented an increase in macrophages, lymphocytes, and neutrophils in bronchoalveolar lavage fluid and MAC2-positive macrophages in lung tissue and peribronchovascular area that was comparable to that observed in WT mice. Conversely, elastase instilled VAChT KDHOM mice showed significantly larger number of NF-κB-positive cells and isoprostane staining in the peribronchovascular area when compared to elastase-instilled WT-mice. Moreover, elastase-instilled VAChT-deficient mice showed increased MCP-1 levels in the lungs. Other cytokines, extracellular matrix remodeling, alveolar enlargement, and lung function were not worse in elastase-instilled VAChT deficiency than in elastase-instilled WT-controls. These data suggest that decreased VAChT expression may contribute to the pathogenesis of emphysema, at least in part, through NF-κB activation, MCP-1, and oxidative stress pathways. This study highlights novel pathways involved in lung inflammation that may contribute to the development of chronic obstrutive lung disease (COPD) in cholinergic deficient individuals such as Alzheimer's disease patients.

Lung Edema and Mortality Induced by Intestinal Ischemia and Reperfusion Is Regulated by VAChT Levels in Female Mice.

Acute lung injury induced by intestinal ischemia/reperfusion (I/R) is a relevant clinical condition. Acetylcholine (ACh) and the α7 nicotinic ACh receptor (nAChRα-7) are involved in the control of inflammation. Mice with reduced levels of the vesicular ACh transporter (VAChT), a protein responsible for controlling ACh release, were used to test the involvement of cholinergic signaling in lung inflammation due to intestinal I/R. Female mice with reduced levels of VAChT (VAChT-KDHOM) or wild-type littermate controls (WT) were submitted to intestinal I/R followed by 2 h of reperfusion. Mortality, vascular permeability, and recruitment of inflammatory cells into the lung were investigated. Parts of mice were submitted to ovariectomy (OVx) to study the effect of sex hormones or treated with PNU-282,987 (nAChRα-7 agonist). A total of 43.4% of VAChT-KDHOM-I/R mice died in the reperfusion period compared to 5.2% of WT I/R mice. The I/R increased lung inflammation in both genotypes. In VAChT-KDHOM mice, I/R increased vascular permeability and decreased the release of cytokines in the lung compared to WT I/R mice. Ovariectomy reduced lung inflammation and permeability compared to non-OVx, but it did not avoid mortality in VAChT-KDHOM-I/R mice. PNU treatment reduced lung permeability, increased the release of proinflammatory cytokines and the myeloperoxidase activity in the lungs, and prevented the increased mortality observed in VAChT-KDHOM mice. Cholinergic signaling is an important component of the lung protector response against intestinal I/R injury. Decreased cholinergic signaling seems to increase pulmonary edema and dysfunctional cytokine release that increased mortality, which can be prevented by increasing activation of nAChRα-7.

Increased cholinergic activity under conditions of low estrogen leads to adverse cardiac remodeling.

Cholinesterase inhibitors are used in postmenopausal women for the treatment of neurodegenerative diseases. Despite their widespread use in the clinical practice, little is known about the impact of augmented cholinergic signaling on cardiac function under reduced estrogen conditions. To address this gap, we subjected a genetically engineered murine model of systemic vesicular acetylcholine transporter overexpression (Chat-ChR2) to ovariectomy and evaluated cardiac parameters. Left-ventricular function was similar between Chat-ChR2 and wild-type (WT) mice. Following ovariectomy, WT mice showed signs of cardiac hypertrophy. Conversely, ovariectomized (OVX) Chat-ChR2 mice evolved to cardiac dilation and failure. Transcript levels for cardiac stress markers atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) were similarly upregulated in WT/OVX and Chat-ChR2/OVX mice. 17ß-Estradiol (E2) treatment normalized cardiac parameters in Chat-ChR2/OVX to the Chat-ChR2/SHAM levels, providing a link between E2 status and the aggravated cardiac response in this model. To investigate the cellular basis underlying the cardiac alterations, ventricular myocytes were isolated and their cellular area and contractility were assessed. Myocytes from WT/OVX mice were wider than WT/SHAM, an indicative of concentric hypertrophy, but their fractional shortening was similar. Conversely, Chat-ChR2/OVX myocytes were elongated and presented contractile dysfunction. E2 treatment again prevented the structural and functional changes in Chat-ChR2/OVX myocytes. We conclude that hypercholinergic mice under reduced estrogen conditions do not develop concentric hypertrophy, a critical compensatory adaptation, evolving toward cardiac dilation and failure. This study emphasizes the importance of understanding the consequences of cholinesterase inhibition, used clinically to treat dementia, for cardiac function in postmenopausal women.

Information Processing in the Brain as Optimal Entropy Transport: A Theoretical Approach.

We consider brain activity from an information theoretic perspective. We analyze the information processing in the brain, considering the optimality of Shannon entropy transport using the Monge-Kantorovich framework. It is proposed that some of these processes satisfy an optimal transport of informational entropy condition. This optimality condition allows us to derive an equation of the Monge-Ampère type for the information flow that accounts for the branching structure of neurons via the linearization of this equation. Based on this fact, we discuss a version of Murray's law in this context.

Effects of VAChT reduction and α7nAChR stimulation by PNU-282987 in lung inflammation in a model of chronic allergic airway inflammation.

The cholinergic anti-inflammatory pathway has been shown to regulate lung inflammation and cytokine release in acute models of inflammation, mainly via α7 nicotinic receptor (α7nAChR). We aimed to evaluate the role of endogenous acetylcholine in chronic allergic airway inflammation in mice and the effects of therapeutic nAChR stimulation in this model. We first evaluated lung inflammation and remodeling on knock-down mice with 65% of vesicular acetylcholine transport (VAChT) gene reduction (KDVAChT) and wild-type(WT) controls that were subcutaneously sensitized and then inhaled with ovalbumin(OVA). We then evaluated the effects of PNU-282987(0.5-to-2mg/kg),(α7nAChR agonist) treatment in BALB/c male mice intraperitoneal sensitized and then inhaled with OVA. Another OVA-sensitized-group was treated with PNU-282987 plus Methyllycaconitine (MLA,1 mg/kg, α7nAChR antagonist) to confirm that the effects observed by PNU were due to α7nAChR. We showed that KDVAChT-OVA mice exhibit exacerbated airway inflammation when compared to WT-OVA mice. In BALB/c, PNU-282987 treatment reduced the number of eosinophils in the blood, BAL fluid, and around airways, and also decreased pulmonary levels of IL-4,IL-13,IL-17, and IgE in the serum of OVA-exposed mice. MLA pre-treatment abolished all the effects of PNU-282987. Additionally, we showed that PNU-282987 inhibited STAT3-phosphorylation and reduced SOCS3 expression in the lung. These data indicate that endogenous cholinergic tone is important to control allergic airway inflammation in a murine model. Moreover, α7nAChR is involved in the control of eosinophilic inflammation and airway remodeling, possibly via inhibition of STAT3/SOCS3 pathways. Together these data suggest that cholinergic anti-inflammatory system mainly α7nAChR should be further considered as a therapeutic target in asthma.

Evaluation of the neuromuscular junction in a middle-aged mouse model of congenital myasthenic syndrome.

INTRODUCTION: Reduced expression of the vesicular acetylcholine transporter (VAChT) leads to changes in the distribution and shape of synaptic vesicles (SVs) at neuromuscular junctions (NMJs), suggesting vesicular acetylcholine (ACh) as a key component of synaptic structure and function. It is poorly understood how long-term changes in cholinergic transmission contribute to age- and disease-related degeneration in the motor system. METHODS: In this study we performed confocal imaging, electrophysiology, electron microscopy, and analyses of respiratory mechanics of the diaphragm NMJ components in 12-month-old wild-type (WT) and VAChTKDHOM mice. RESULTS: Diaphragms of NMJs of the VAChTKDHOM mice were similar to those in WT mice in number, colocalization, and fragmentation of pre-/postsynaptic components. However, they had increased spontaneous SV exocytosis, miniature endplate potential frequency, and diminished MEPP amplitude. No impairment in respiratory mechanics at rest was observed, probably due to the large neurotransmission safety factor of the diaphragm. DISCUSSION: The present findings help us to understand the consequences of reduced ACh release at the NMJs during aging.

Vesicular acetylcholine transport deficiency potentiates some inflammatory responses induced by diesel exhaust particles.

Endogenous acetylcholine (ACh), which depends of the levels of vesicular ACh transport (VAChT) to be released, is the central mediator of the cholinergic anti-inflammatory system. ACh controls the release of cytokine in different models of inflammation. Diesel exhaust particles (DEP) are one of the major environmental pollutants produced in large quantity by automotive engines in urban center. DEP bind the lung parenchyma and induce inflammation. We evaluated whether cholinergic dysfunction worsens DEP-induced lung inflammation. Male mice with decreased ACh release due to reduced expression of VAChT (VAChT-KD mice) were submitted to DEP exposure for 30 days (3 mg/mL of DEP, once a day, five days a week) or saline. Pulmonary function and inflammation as well as extracellular matrix fiber deposition were evaluated. Additionally, airway and nasal epithelial mucus production were quantified. We found that DEP instillation worsened lung function and increased lung inflammation. Higher levels of mononuclear cells were observed in the peripheral blood of both wild-type (WT) and VAChT-KD mice. Also, both wild-type (WT) and VAChT-KD mice showed an increase in macrophages in bronchoalveolar lavage fluid (BALF) as well as increased expression of IL-4, IL-6, IL-13, TNF-α, and NF-κB in lung cells. The collagen fiber content in alveolar septa was also increased in both genotypes. On the other hand, we observed that granulocytes were increased only in VAChT-KD peripheral blood. Likewise, increased BALF lymphocytes and neutrophils as well as increased elastic fibers in alveolar septa, airway neutral mucus, and nasal epithelia acid mucus were observed only in VAChT-KD mice. The cytokines IL-4 and TNF-α were also higher in VAChT-KD mice compared with WT mice. In conclusion, decreased ability to release ACh exacerbates some of the lung alterations induced by DEP in mice, suggesting that VAChT-KD animals are more vulnerable to the effects of DEP in the lung.

Acute lung injury is reduced by the α7nAChR agonist PNU-282987 through changes in the macrophage profile.

Nicotinic α-7 acetylcholine receptor (nAChRα7) is a critical regulator of cholinergic anti-inflammatory actions in several diseases, including acute respiratory distress syndrome (ARDS). Given the potential importance of α7nAChR as a therapeutic target, we evaluated whether PNU-282987, an α7nAChR agonist, is effective in protecting the lung against inflammation. We performed intratracheal instillation of LPS to generate acute lung injury (ALI) in C57BL/6 mice. PNU-282987 treatment, either before or after ALI induction, reduced neutrophil recruitment and IL-1ß, TNF-α, IL-6, keratinocyte chemoattractant (KC), and IL-10 cytokine levels in the bronchoalveolar lavage fluid (P < 0.05). In addition, lung NF-κB phosphorylation decreased, along with collagen fiber deposition and the number of matrix metalloproteinase-9+ and -2+ cells, whereas the number of tissue inhibitor of metalloproteinase-1+ cells increased (P < 0.05). PNU-282987 treatment also reduced lung mRNA levels and the frequency of M1 macrophages, whereas cells expressing the M2-related markers CD206 and IL-10 increased, suggesting changes in the macrophage profile. Finally, PNU-282987 improved lung function in LPS-treated animals. The collective results suggest that PNU-282987, an agonist of α7nAChR, reduces LPS-induced experimental ALI, thus supporting the notion that drugs that act on α7nAChRs should be explored for ARDS treatment in humans.-Pinheiro, N. M., Santana, F. P. R., Almeida, R. R., Guerreiro, M., Martins, M. A., Caperuto, L. C., Câmara, N. O. S., Wensing, L. A., Prado, V. F., Tibério, I. F. L. C., Prado, M. A. M., Prado, C. M. Acute lung injury is reduced by the α7nAChR agonist PNU-282987 through changes in the macrophage profile.

Reduced expression of VAChT increases renal fibrosis.

Chronic kidney disease (CKD) is associated with several other long-lasting conditions such as diabetes and cardiovascular diseases and it is a significant contributor to mortality worldwide. Obstructive kidney disease is one of the leading causes of CKD in children and may result from a wide variety of pathologic processes. Recent studies have shown that α7 nicotinic acetylcholine receptor (α7 nAChR) activation in the cholinergic anti-inflammatory pathway reduces production of inflammatory mediators and consequently prevents tissue injury and death. Here, we examined the role of endogenous release of acetylcholine on the development of fibrosis in renal tissue using a model of unilateral ureter obstruction (UUO)-induced CKD, in which obstruction promotes inflammation-mediated kidney damages. To interfere with acetylcholine secretion, we used mice in which the vesicular acetylcholine transporter is genetically reduced (VAChT KD(hom) mice). We observed a higher renal damage in VAChT mutant mice when compared to wild type controls, exemplified by higher proteinuria and increased amount of type 1 collagen in the kidney tissue, indicating accentuated fibrogenesis. These results were accompanied by enhanced localized kidney inflammation, with increased TH1/TH17 profile response. Administration of PNU-282987, a selective agonist of α7 nAChR, significantly attenuated kidney injury after UUO in VAChT KD(hom) mice, indicating that the lack of acetylcholine release decrease the action of the cholinergic anti-inflammatory pathway, promoting an up-regulation of pro-inflammatory and pro-fibrotic pathways. These results suggest that physiological activation of the cholinergic anti-inflammatory pathway regulates inflammatory responses in the kidney suggesting a new therapeutic approach for kidney disease.

Vesicular acetylcholine transporter knock down-mice are more susceptible to inflammation, c-Fos expression and sickness behavior induced by lipopolysaccharide.

In addition to the well-known functions as a neurotransmitter, acetylcholine (ACh) can modulate of the immune system. Nonetheless, how endogenous ACh release inflammatory responses is still not clear. To address this question, we took advantage of an animal model with a decreased ACh release due a reduction (knockdown) in vesicular acetylcholine transporter (VAChT) expression (VAChT-KD(HOM)). These animals were challenged with lipopolysaccharide (LPS). Afterwards, we evaluated sickness behavior and quantified systemic and cerebral inflammation as well as neuronal activation in the dorsal vagal complex (DVC). VAChT-KD(HOM) mice that were injected with LPS (10mg/kg) showed increased mortality rate as compared to control mice. In line with this result, a low dose of LPS (0.1mg/kg) increased the levels of pro-inflammatory (TNF-α, IL-1ß, and IL-6) and anti-inflammatory (IL-10) cytokines in the spleen and brain of VAChT-KD(HOM) mice in comparison with controls. Similarly, serum levels of TNF-α and IL-6 were increased in VAChT-KD(HOM) mice. This excessive cytokine production was completely prevented by administration of a nicotinic receptor agonist (0.4mg/kg) prior to the LPS injection. Three hours after the LPS injection, c-Fos expression increased in the DVC region of VAChT-KD(HOM) mice compared to controls. In addition, VAChT-KD(HOM) mice showed behavioral changes such as lowered locomotor and exploratory activity and reduced social interaction after the LPS challenge, when compared to control mice. Taken together, our results show that the decreased ability to release ACh exacerbates systemic and cerebral inflammation and promotes neural activation and behavioral changes induced by LPS. In conclusion, our findings support the notion that activity of cholinergic pathways, which can be modulated by VAChT expression, controls inflammatory and neural responses to LPS challenge.

Endogenous Acetylcholine Controls the Severity of Polymicrobial Sepsisassociated Inflammatory Response in Mice.

Acetylcholine (ACh) is the main mediator associated with the anti-inflammatory cholinergic pathway. ACh plays an inhibitory role in several inflammatory conditions. Sepsis is a severe clinical syndrome characterized by bacterial dissemination and overproduction of inflammatory mediators. The aim of the current study was to investigate the participation of endogenous ACh in the modulation of inflammatory response induced by a model of polymicrobial sepsis. Wild type (WT) and vesicular acetylcholine transporter knockdown (VAChT(KD)) mice were exposed to cecal ligation and perforation- induced sepsis. Levels of Tumor Necrosis Factor Alpha (TNF-α) and bacterial growth in peritoneal cavity and serum, and neutrophil recruitment into peritoneal cavity were assessed. The concentration of TNF-α in both compartments was higher in VAChT(KD) in comparison with WT mice. VAChT(KD) mice presented elevated burden of bacteria in peritoneum and blood, and impairment of neutrophil migration to peritoneal cavity. This phenotype was reversed by treatment with nicotine salt. These findings suggest that endogenous ACh plays a major role in the control of sepsis-associated inflammatory response.

Increased airway reactivity and hyperinsulinemia in obese mice are linked by ERK signaling in brain stem cholinergic neurons.

Obesity is a major risk factor for asthma, which is characterized by airway hyperreactivity (AHR). In obesity-associated asthma, AHR may be regulated by non-TH2 mechanisms. We hypothesized that airway reactivity is regulated by insulin in the CNS, and that the high levels of insulin associated with obesity contribute to AHR. We found that intracerebroventricular (ICV)-injected insulin increases airway reactivity in wild-type, but not in vesicle acetylcholine transporter knockdown (VAChT KD(HOM-/-)), mice. Either neutralization of central insulin or inhibition of extracellular signal-regulated kinases (ERK) normalized airway reactivity in hyperinsulinemic obese mice. These effects were mediated by insulin in cholinergic nerves located at the dorsal motor nucleus of the vagus (DMV) and nucleus ambiguus (NA), which convey parasympathetic outflow to the lungs. We propose that increased insulin-induced activation of ERK in parasympathetic pre-ganglionic nerves contributes to AHR in obese mice, suggesting a drug-treatable link between obesity and asthma.

Pulmonary inflammation is regulated by the levels of the vesicular acetylcholine transporter.

Acetylcholine (ACh) plays a crucial role in physiological responses of both the central and the peripheral nervous system. Moreover, ACh was described as an anti-inflammatory mediator involved in the suppression of exacerbated innate response and cytokine release in various organs. However, the specific contributions of endogenous release ACh for inflammatory responses in the lung are not well understood. To address this question we have used mice with reduced levels of the vesicular acetylcholine transporter (VAChT), a protein required for ACh storage in secretory vesicles. VAChT deficiency induced airway inflammation with enhanced TNF-α and IL-4 content, but not IL-6, IL-13 and IL-10 quantified by ELISA. Mice with decreased levels of VAChT presented increased collagen and elastic fibers deposition in airway walls which was consistent with an increase in inflammatory cells positive to MMP-9 and TIMP-1 in the lung. In vivo lung function evaluation showed airway hyperresponsiveness to methacholine in mutant mice. The expression of nuclear factor-kappa B (p65-NF-kB) in lung of VAChT-deficient mice were higher than in wild-type mice, whereas a decreased expression of janus-kinase 2 (JAK2) was observed in the lung of mutant animals. Our findings show the first evidence that cholinergic deficiency impaired lung function and produce local inflammation. Our data supports the notion that cholinergic system modulates airway inflammation by modulation of JAK2 and NF-kB pathway. We proposed that intact cholinergic pathway is necessary to maintain the lung homeostasis.

Nitric oxide regulates AKT phosphorylation and nuclear translocation in cultured retinal cells.

Previous studies have shown that nitric oxide (NO) inhibits apoptosis of retinal neurons in culture through the canonical cyclic GMP/protein kinase G (PKG)-dependent pathway, but also involving multiple kinase pathways, such as phosphatidylinositol 3' kinase (PI3k) and AKT. NO and AKT exhibit survival-promoting properties and display important roles in both CNS development and plasticity. The purpose of this study was to evaluate the effects of exogenous NO, derived from the NO donor S-nitroso-N-acetylpenicillamin (SNAP), or endogenous NO, produced from l-arginine, on AKT phosphorylation in cultured chick retinal neurons. Our results demonstrate that SNAP or l-arginine enhances AKT phosphorylation on both serine-473 and threonine-308 residues in a concentration and time-dependent manner. This effect was mediated by the activation of soluble guanylyl cyclase and PKG, since it was blocked by the respective enzyme inhibitors ODQ or LY83583 and KT5823, as well as by transduction with shRNA lentiviruses coding PKGII shRNA, and mimicked by the respective enzyme activators YC-1 and 8-Bromo cyclic GMP, and also by the cyclic GMP phosphodiesterase inhibitor zaprinast. In addition, LY294002 or wortmannin suppressed the SNAP effect, indicating the involvement of phosphoinositide 3' kinase. Moreover, the mTOR inhibitor KU0063794 blocked SNAP-induced AKT phosphorylation at both residues, suggesting the participation of the mTORC2 complex in the process. Glutamate and NMDA also promoted AKT phosphorylation and a nitric oxide synthase inhibitor abrogated these effects, revealing a mechanism involving the activation of NMDA receptors and NO production. We have also found that SNAP and l-arginine induced AKT translocation into the nucleus of retinal neurons as well as other neuronal cell lines. SNAP also protects retinal cells from death induced by hydrogen peroxide and this effect was blocked by the phosphoinositide 3' kinase inhibitor LY294002. We therefore conclude that NO produced from endogenous or exogenous sources promotes AKT activation and its shuttling to the nucleus, probably participating in neuronal survival pathways important during CNS development.

Anaerobic digestion of wastewater from coffee and chemical analysis of biogas produced using gas chromatography: quantification of methane, and potential energy gas exchanger / Digestão anaeróbia de águas residuárias do café e análise química do biogás produzido por cromatografia gasosa: quantificação de metano e potencial de troca gasosa de energia

Methane is the main constituent of biogas, being responsible for its calorific value. This work objective was to present the analysis of methane concentration in the biogas originated from anaerobic treatment system of wastewater of coffee wet processing (CWP) at laboratory scale, using coffee coconut. The methane concentration were performed by gas-solid chromatography (GSC) analyses. The system was composed of a column Restek RT-Q-PLOT, with the stage sets, the divinyl-benzene, nitrogen as mobile phase and a flame ionization detector (FID). The results of the concentration of methane in the biogas ranged from 48.60 to 68.14%. The upper and lower calorific values were 25,654 and 23,777 kJ.m-3, respectively. For interchangeability, obtained a Wobbe number of 7,851 kcal.m-3, resulting in their interchangeability with piped gas (city gas).

O metano é o principal constituinte do biogás, sendo o responsável pelo seu poder calorífico. Neste trabalho, são apresentadas as análises da concentração de metano no biogás produzido a partir do tratamento das águas residuárias do processamento por via úmida do café (ARC) em sistema de tratamento anaeróbio em escala de laboratório, sendo utilizado o café coco para a produção destas ARC. As análises foram realizadas por cromatografia gás-sólido (CGS), sendo o sistema composto de uma coluna Restek RT-Q-PLOT, tendo como fase fixa, o divinil-benzeno; do nitrogênio como fase móvel e um detector de ionização de chama (DIC). Os resultados da concentração de metano no biogás variaram de 48,60 a 68,14 %, sendo estas variações obtidas em função dos parâmetros do processo de tratamento. Os poderes caloríficos superior e inferior foram de 25.654 e 23.777 kJ.m-3, respectivamente. Para a permutabilidade, obteve-se um número de Wobbe de 7.851 kcal.m-3, resultando na sua permutabilidade com o gás canalizado (gás da cidade).

Autonomic nervous system modulation affects the inflammatory immune response in mice with acute Chagas disease.

The aim of the present study was to evaluate the effects of changes to the autonomic nervous system in mice during the acute phase of Chagas disease, which is an infection caused by the parasite Trypanosoma cruzi. The following types of mice were inoculated with T. cruzi (CHG): wild-type (WT) and vesicular acetylcholine transporter knockdown (KDVAChT) C57BL/6j mice; wild-type non-treated (NT) FVB mice; FVB mice treated with pyridostigmine bromide (PYR) or salbutamol (SALB); and ß(2)-adrenergic receptor knockout (KOß2) FVB mice. During infection and at 18-21 days after infection (acute phase), the survival curves, parasitaemia, electrocardiograms, heart rate variability, autonomic tonus and histopathology of the animals were evaluated. Negative control groups were matched for age, genetic background and treatment. The KDVAChT-CHG mice exhibited a significant shift in the electrocardiographic, autonomic and histopathological profiles towards a greater inflammatory immune response that was associated with a reduction in blood and tissue parasitism. In contrast, the CHG-PYR mice manifested reduced myocardial inflammation and lower blood and tissue parasitism. Similar results were observed in CHG-SALB animals. Unexpectedly, the KOß2-CHG mice exhibited less myocardial inflammation and higher blood and tissue parasitism, which were associated with reduced mortality. These findings could have been due to the increase in vagal tone observed in the KOß2 mice, which rendered them more similar to the CHG-PYR animals. In conclusion, our results indicate a marked immunomodulatory role for the parasympathetic and sympathetic autonomic nervous systems, which inhibit both the inflammatory immune response and parasite clearance during the acute phase of experimental Chagas heart disease in mice.

Antinociceptive effect of Brazilian armed spider venom toxin Tx3-3 in animal models of neuropathic pain.

Venoms peptides have produced exceptional sources for drug development to treat pain. In this study we examined the antinociceptive and side effects of Tx3-3, a peptide toxin isolated from Phoneutria nigriventer venom, which inhibits high-voltage-dependent calcium channels (VDCC), preferentially P/Q and R-type VDCC. We tested the effects of Tx3-3 in animal models of nociceptive (tail-flick test), neuropathic (partial sciatic nerve ligation and streptozotocin-induced diabetic neuropathy), and inflammatory (intraplantar complete Freund's adjuvant) pain. In the tail-flick test, both intrathecal (i.t.) and intracerebroventricular (i.c.v.) injection of Tx3-3 in mice caused a short-lasting effect (ED(50) and 95% confidence intervals of 8.8 [4.1-18.8] and 3.7 [1.6-8.4] pmol/site for i.t. and i.c.v. injection, respectively), without impairing motor functions, at least at doses 10-30 times higher than the effective dose. By comparison, ω-conotoxin MVIIC, a P/Q and N-type VDCC blocker derived from Conus magus venom, caused significant motor impairment at doses close to efficacious dose in tail flick test. Tx3-3 showed a long-lasting antinociceptive effect in neuropathic pain models. Intrathecal injection of Tx3-3 (30 pmol/site) decreased both mechanical allodynia produced by sciatic nerve injury in mice and streptozotocin-induced allodynia in mice and rats. On the other hand, i.t. injection of Tx3-3 did not alter inflammatory pain. Taken together, our data show that Tx3-3 shows prevalent antinociceptive effects in the neuropathic pain models and does not cause adverse motor effects at antinociceptive efficacious doses, suggesting that this peptide toxin holds promise as a novel therapeutic agent for the control of neuropathic pain. The Brazilian armed spider Tx3-3, a new P/Q and R-type calcium channel blocker, effectively alleviates allodynia in animal neuropathic pain models.