<Emphasis Type="Italic">Gad67</Emphasis> haploinsufficiency reduces amyloid pathology and rescues olfactory memory deficits in a mouse model of Alzheimer’s disease

Background

Alzheimer’s disease (AD) is the most common age-related neurodegenerative disorder, affecting millions of people worldwide. Although dysfunction of multiple neurotransmitter systems including cholinergic, glutamatergic and GABAergic systems has been associated with AD progression the underlying mechanisms remain elusive. We and others have recently found that GABA content is elevated in AD brains and linked to cognitive deficits in AD mouse models. The glutamic acid decarboxylase 67 (GAD67) is the major enzyme converting glutamate into GABA and has been implied in a number of neurological disorders such as epilepsy and schizophrenia. However, whether Gad67 is involved in AD pathology has not been well studied. Here, we investigate the functional role of GAD67 in an AD mouse model with Gad67 haploinsufficiency that is caused by replacing one allele of Gad67 with green fluorescent protein (GFP) gene during generation of GAD67-GFP mice.

Methods

To genetically reduce GAD67 in AD mouse brains, we crossed the Gad67 haploinsufficient mice (GAD67-GFP^+/?) with 5xFAD mice (harboring 5 human familial AD mutations in APP and PS1 genes) to generate a new line of bigenic mice. Immunostaining, ELISA, electrophysiology and behavior test were applied to compare the difference between groups.

Results

We found that reduction of GAD67 resulted in a significant decrease of amyloid β production in 5xFAD mice. Concurrently, the abnormal astrocytic GABA and tonic GABA currents, as well as the microglial reactivity were significantly reduced in the 5xFAD mice with Gad67 haploinsufficiency. Importantly, the olfactory memory deficit of 5xFAD mice was rescued by Gad67 haploinsufficiency.

Conclusions

Our results demonstrate that GAD67 plays an important role in AD pathology, suggesting that GAD67 may be a potential drug target for modulating the progress of AD.

     

Roles of Bronchopulmonary C-fibers in airway Hyperresponsiveness and airway remodeling induced by house dust mite

Background

Asthma is characterized by chronic airway inflammation, airway hyperresponsiveness (AHR), and airway remodeling. While exposure of house dust mites (HDM) is a common cause of asthma, the pathogenesis of the HDM-induced asthma is not fully understood. Bronchopulmonary C-fibers (PCFs) contribute to the neurogenic inflammation, viral infection induced-persistent AHR, and ovalbumin induced collagen deposition largely via releasing neuropeptides, such as substance P (SP). However, PCF roles in the pathogenesis of the HDM-induced asthma remain unexplored. The goal of this study was to determine what role PCFs played in generating these characteristics.

Methods

We compared the following variables among the PCF-intact and -degenerated BALB/c mice with and without chronic HDM exposure (four groups): 1) AHR and pulmonary SP; 2) airway smooth muscle (ASM) mass; 3) pulmonary inflammatory cells; and 4) epithelium thickening and mucus secretion.

Results

We found that HDM evoked AHR associated with upregulation of pulmonary SP and inflammation, ASM mass increase, epithelium thickenings, and mucus hypersecretion. PCF degeneration decreased the HDM-induced changes in AHR, pulmonary SP and inflammation, and ASM mass, but failed to significantly affect the epithelium thickening and mucus hypersecretion.

Conclusion

Our data suggest an involvement of PCFs in the mechanisms by which HDM induces allergic asthma via airway inflammation, AHR, and airway remodeling.

     

Prolonged ozone exposure in an allergic airway disease model: Adaptation of airway responsiveness and airway remodeling

Background

Short-term exposure to high concentrations of ozone has been shown to increase airway hyper-responsiveness (AHR). Because the changes in AHR and airway inflammation and structure after chronic ozone exposure need to be determined, the goal of this study was to investigate these effects in a murine model of allergic airway disease.

Methods

We exposed BALB/c mice to 2 ppm ozone for 4, 8, and 12 weeks. We measured the enhanced pause (Penh) to methacholine and performed cell differentials in bronchoalveolar lavage fluid. We quantified the levels of IL-4 and IFN-γ in the supernatants of the bronchoalveolar lavage fluids using enzyme immunoassays, and examined the airway architecture under light and electron microscopy.

Results

The groups exposed to ozone for 4, 8, and 12 weeks demonstrated decreased Penh at methacholine concentrations of 12.5, 25, and 50 mg/ml, with a dose-response curve to the right of that for the filtered-air group. Neutrophils and eosinophils increased in the group exposed to ozone for 4 weeks compared to those in the filtered-air group. The ratio of IL-4 to INF-γ increased significantly after exposure to ozone for 8 and 12 weeks compared to the ratio for the filtered-air group. The numbers of goblet cells, myofibroblasts, and smooth muscle cells showed time-dependent increases in lung tissue sections from the groups exposed to ozone for 4, 8, and 12 weeks.

Conclusion

These findings demonstrate that the increase in AHR associated with the allergic airway does not persist during chronic ozone exposure, indicating that airway remodeling and adaptation following repeated exposure to air pollutants can provide protection against AHR.

     

The effect of 1,25-dihydroxyvitamin D<Subscript>3</Subscript> on TSLP,IL-33 and IL-25 expression in respiratory epithelium

Background

Airway epithelium is an active and important component of the immunological response in the pathophysiology of obstructive lung diseases. Recent studies suggest an important role for vitamin D₃ in asthma severity and treatment response.

Objective

Our study evaluated the influence of an active form of vitamin D₃ on the expression of selected mediators of allergic inflammation in the respiratory epithelium.

Material and Methods

Primary nasal and bronchial epithelial cells were exposed to1,25D₃ for 1 hour and were then stimulated or not with IL-4, TNF-α, LPS, and poly I:C. After 24 hours TSLP, IL-33, and IL-25 protein levels were measured in culture supernatants usingELISAandmRNAlevels in cells by real time PCR.

Results

1,25D₃ increased TSLP concentration in unstimulated nasal epithelial cells, but did not influence IL-33 and IL-25 expression. In IL-4-stimulated epithelial cell cultures 1,25D₃ mostly inhibited TSLP and IL-33 expression. In LPS-treated cultures 1,25D₃ decreased IL-33 expression. Simultaneously 1,25D₃ augmented IL-25 production in the same model of stimulation.

Conclusion

Our study revealed the dual nature of vitamin D₃ manifested in both pro- and anti-inflammatory properties observed in airway epithelial cells.

     

Pharmacological characterization of GABA<Subscript>A</Subscript> receptors in taurine-fed mice

Background

Taurine is one of the most abundant free amino acids especially in excitable tissues, with wide physiological actions. Chronic supplementation of taurine in drinking water to mice increases brain excitability mainly through alterations in the inhibitory GABAergic system. These changes include elevated expression level of glutamic acid decarboxylase (GAD) and increased levels of GABA. Additionally we reported that GABA_A receptors were down regulated with chronic administration of taurine. Here, we investigated pharmacologically the functional significance of decreased / or change in subunit composition of the GABA_A receptors by determining the threshold for picrotoxin-induced seizures. Picrotoxin, an antagonist of GABA_A receptors that blocks the channels while in the open state, binds within the pore of the channel between the β2 and β3 subunits. These are the same subunits to which GABA and presumably taurine binds.

Methods

Two-month-old male FVB/NJ mice were subcutaneously injected with picrotoxin (5 mg kg^-1) and observed for a) latency until seizures began, b) duration of seizures, and c) frequency of seizures. For taurine treatment, mice were either fed taurine in drinking water (0.05%) or injected (43 mg/kg) 15 min prior to picrotoxin injection.

Results

We found that taurine-fed mice are resistant to picrotoxin-induced seizures when compared to age-matched controls, as measured by increased latency to seizure, decreased occurrence of seizures and reduced mortality rate. In the picrotoxin-treated animals, latency and duration were significantly shorter than in taurine-treated animas. Injection of taurine 15 min before picrotoxin significantly delayed seizure onset, as did chronic administration of taurine in the diet. Further, taurine treatment significantly increased survival rates compared to the picrotoxin-treated mice.

Conclusions

We suggest that the elevated threshold for picrotoxin-induced seizures in taurine-fed mice is due to the reduced binding sites available for picrotoxin binding due to the reduced expression of the beta subunits of the GABA_A receptor. The delayed effects of picrotoxin after acute taurine injection may indicate that the two molecules are competing for the same binding site on the GABA_A receptor. Thus, taurine-fed mice have a functional alteration in the GABAergic system. These include: increased GAD expression, increased GABA levels, and changes in subunit composition of the GABA_A receptors. Such a finding is relevant in conditions where agonists of GABA_A receptors, such as anesthetics, are administered.

     

Statistical analysis of fractionation resistance by functional category and expression

Background

The current literature establishes the importance of gene functional category and expression in promoting or suppressing duplicate gene loss after whole genome doubling in plants, a process known as fractionation. Inspired by studies that have reported gene expression to be the dominating factor in preventing duplicate gene loss, we analyzed the relative effect of functional category and expression.

Methods

We use multivariate methods to study data sets on gene retention, function and expression in rosids and asterids to estimate effects and assess their interaction.

Results

Our results suggest that the effect on duplicate gene retention fractionation by functional category and expression are independent and have no statistical interaction.

Conclusion

In plants, functional category is the more dominant factor in explaining duplicate gene loss.

     

Characterization of pediatric cystic fibrosis airway epithelial cell cultures at the air-liquid interface obtained by non-invasive nasal cytology brush sampling

Background

In vitro systems of primary cystic fibrosis (CF) airway epithelial cells are an important tool to study molecular and functional features of the native respiratory epithelium. However, undifferentiated CF airway cell cultures grown under submerged conditions do not appropriately represent the physiological situation. A more advanced CF cell culture system based on airway epithelial cells grown at the air-liquid interface (ALI) recapitulates most of the in vivo-like properties but requires the use of invasive sampling methods. In this study, we describe a detailed characterization of fully differentiated primary CF airway epithelial cells obtained by non-invasive nasal brushing of pediatric patients.

Methods

Differentiated cell cultures were evaluated with immunolabelling of markers for ciliated, mucus-secreting and basal cells, and tight junction and CFTR proteins. Epithelial morphology and ultrastructure was examined by histology and transmission electron microscopy. Ciliary beat frequency was investigated by a video-microscopy approach and trans-epithelial electrical resistance was assessed with an epithelial Volt-Ohm meter system. Finally, epithelial permeability was analysed by using a cell layer integrity test and baseline cytokine levels where measured by an enzyme-linked immunosorbent assay.

Results

Pediatric CF nasal cultures grown at the ALI showed a differentiation into a pseudostratified epithelium with a mucociliary phenotype. Also, immunofluorescence analysis revealed the presence of ciliated, mucus-secreting and basal cells and tight junctions. CFTR protein expression was observed in CF (F508del/F508del) and healthy cultures and baseline interleukin (IL)-8 and IL-6 release were similar in control and CF ALI cultures. The ciliary beat frequency was 9.67 Hz and the differentiated pediatric CF epithelium was found to be functionally tight.

Conclusion

In summary, primary pediatric CF nasal epithelial cell cultures grown at the ALI showed full differentiation into ciliated, mucus-producing and basal cells, which adequately reflect the in vivo properties of the human respiratory epithelium.

     

Proteomic identification of moesin upon exposure to acrolein

Background

Acrolein (allyl Aldehyde) as one of smoke irritant exacerbates chronic airway diseases and increased in sputum of patients with asthma and chronic obstructive lung disease. But underlying mechanism remains unresolved. The aim of study was to identify protein expression in human lung microvascular endothelial cells (HMVEC-L) exposed to acrolein.

Methods

A proteomic approach was used to determine the different expression of proteins at 8 h and 24 h after treatment of acrolein 30 nM and 300 nM to HMVEC-L. Treatment of HMVEC-L with acrolein 30 nM and 300 nM altered 21 protein spots on the two-dimensional gel, and these were then analyzed by MALDI-TOF MS.

Results

These proteins included antioxidant, signal transduction, cytoskeleton, protein transduction, catalytic reduction. The proteins were classified into four groups according to the time course of their expression patterns such as continually increasing, transient increasing, transient decreasing, and continually decreasing. For validation immunohistochemical staining and Western blotting was performed on lung tissues from acrolein exposed mice. Moesin was expressed in endothelium, epithelium, and inflammatory cells and increased in lung tissues of acrolein exposed mice compared with sham treated mice.

Conclusions

These results indicate that some of proteins may be an important role for airway disease exacerbation caused by acrolein exposure.

     

Neuro-endocrine basis for altered plasma glucose homeostasis in the Fragile X mouse

Background

The fragile X mouse model shows an increase in seizure susceptibility, indicating an involvement of the GABAergic system via an alteration in cellular excitability. In the brain, we have previously described a reduction in GABA_A receptor expression as a likely basis for this susceptibility. In the brains of fragile X mice, this reduction in receptor expression culminates with a concomitant increase in the expression of glutamic acid decarboxylase (GAD), the enzyme responsible for GABA synthesis. Further, voltage-sensitive calcium channel expression is reduced in the pancreas of the fragile X mouse. Since there are considerable similarities in the GABAergic system in the brain and pancreas, we evaluated the protective role of taurine in pancreatic islet development in both wild type (WT) and fragile X mice (KO).

Methods

One-month-old FVB/NJ males or age-matched fmr1-knockout (KO) mice were supplemented with taurine in drinking water (0.05% w/v) for four weeks. Age-matched controls were fed water only for the same duration. At four weeks, mice were sacrificed and pancreases processed for histology and immunohistochemical studies on changes of insulin, glucagon and somatostatin expression. Additional mice were subjected to a glucose tolerance test.

Results

Taurine treatment resulted in a significant increase in the number and size of islets. WT taurine-fed mice, slightly hypoglycemic prior to glucose injection, showed significantly reduced plasma glucose at 30 min post-injection when compared to control mice. KO mice had normal baseline plasma glucose concentration; however, following glucose injection they had higher plasma glucose levels at 30 min when compared to controls. Supplementation of taurine to KO mice resulted in reduced baseline levels of plasma glucose. After glucose injection, the taurine-fed KO mice had reduced plasma glucose at 30 min compared to KO. Concomitant with the increased islets size and glucose tolerance observed in taurine-fed mice there was an increase in insulin, glucagon and somatostatin immunoreactivity in the islets of WT mice. In the KO mice however, insulin levels were not affected whereas glucagon and somatostatin levels were reduced. Exocytosis of these hormones is calcium-dependent, therefore any exacerbation of calcium homeostasis could affect hormone release. We found the expression of the voltage sensitive calcium channels (VSCC) is drastically reduced in the pancreas of fragile X mice.

Conclusions

During early development, the VSCC play an important role in calcium-dependent gene expression. Since these channels are also involved in depolarization and calcium-mediated vesicular release of neurotransmitters and pancreatic hormones, alterations in the expression of VSCC not only will affect calcium-mediated gene expression but also hormonal and neurotransmitter release creating therefore a neuroendocrine perturbation in the fragile X that may potentially affect other organ systems. We find that in the fragile X mouse, taurine treatment may partially restore functionality of the neuro-endocrine pancreas.

     

Population genetic variation in gene expression is associated with phenotypic variation in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Background

The relationship between genetic variation in gene expression and phenotypic variation observable in nature is not well understood. Identifying how many phenotypes are associated with differences in gene expression and how many gene-expression differences are associated with a phenotype is important to understanding the molecular basis and evolution of complex traits.

Results

We compared levels of gene expression among nine natural isolates of Saccharomyces cerevisiae grown either in the presence or absence of copper sulfate. Of the nine strains, two show a reduced growth rate and two others are rust colored in the presence of copper sulfate. We identified 633 genes that show significant differences in expression among strains. Of these genes, 20 were correlated with resistance to copper sulfate and 24 were correlated with rust coloration. The function of these genes in combination with their expression pattern suggests the presence of both correlative and causative expression differences. But the majority of differentially expressed genes were not correlated with either phenotype and showed the same expression pattern both in the presence and absence of copper sulfate. To determine whether these expression differences may contribute to phenotypic variation under other environmental conditions, we examined one phenotype, freeze tolerance, predicted by the differential expression of the aquaporin gene AQY2. We found freeze tolerance is associated with the expression of AQY2.

Conclusions

Gene expression differences provide substantial insight into the molecular basis of naturally occurring traits and can be used to predict environment dependent phenotypic variation.

     

Association between HMGB1 and asthma: a literature review

Background

Recently, some studies demonstrated that HMGB1, as proinflammatory mediator belonging to the alarmin family, has a key role in different acute and chronic immune disorders. Asthma is a complex disease characterised by recurrent and reversible airflow obstruction associated to airway hyper-responsiveness and airway inflammation.

Objective

This literature review aims to analyse advances on HMGB1 role, employment and potential diagnostic application in asthma.

Methods

We reviewed experimental studies that investigated the pathogenetic role of HMGB in bronchial airway hyper-responsiveness, inflammation and the correlation between HMGB1 level and asthma.

Results

A total of 19 studies assessing the association between HMGB1 and asthma were identified.

Conclusions

What emerged from this literature review was the confirmation of HMGB-1 involvement in diseases characterised by chronic inflammation, especially in pulmonary pathologies. Findings reported suggest a potential role of the alarmin in being a stadiation method and a marker of therapeutic efficacy; finally, inhibiting HMGB1 in humans in order to contrast inflammation should be the aim for future further studies.

     

Metabolic response of porcine colon explants to in vitro infection by <Emphasis Type="Italic">Brachyspira hyodysenteriae</Emphasis>: a leap into disease pathophysiology

Introduction

Swine dysentery caused by Brachyspira hyodysenteriae is a production limiting disease in pig farming. Currently antimicrobial therapy is the only treatment and control method available.

Objective

The aim of this study was to characterize the metabolic response of porcine colon explants to infection by B. hyodysenteriae.

Methods

Porcine colon explants exposed to B. hyodysenteriae were analyzed for histopathological, metabolic and pro-inflammatory gene expression changes.

Results

Significant epithelial necrosis, increased levels of l-citrulline and IL-1α were observed on explants infected with B. hyodysenteriae.

Conclusions

The spirochete induces necrosis in vitro likely through an inflammatory process mediated by IL-1α and NO.

     

Neutrophil extracellular traps are downregulated by glucocorticosteroids in lungs in an equine model of asthma

Background

Severe neutrophilic asthma is poorly responsive to glucocorticosteroids (GC). Neutrophil extracellular traps (NETs) within the lungs have been associated with the severity of airway obstruction and inflammation in asthma, and were found to be unaffected by GC in vitro. As IL-17 is overexpressed in neutrophilic asthma and contributes to steroid insensitivity in different cell types, we hypothesized that NETs formation in asthmatic airways would be resistant to GC through an IL-17 mediated pathway.

Methods

Six neutrophilic severe asthmatic horses and six healthy controls were studied while being treated with dexamethasone. Lung function, bronchoalveolar lavage fluid (BALF) cytology and NETs formation, as well as the expression of CD11b and CD13 by blood and airway neutrophils were evaluated. The expression of IL-17 and its role in NETs formation were also studied.

Results

Airway neutrophils from asthmatic horses, as opposed to blood neutrophils, enhanced NETs formation, which was then decreased by GC. GC also tended to decrease the expression of CD11b in blood neutrophils, but not in airway neutrophils. IL-17 mRNA was increased in BALF cells of asthmatic horses and was unaffected by GC. However, both GC and IL-17 inhibited NETs formation in vitro.

Conclusion

GC decreased NETs formation in vitro and also in vivo in the lungs of asthmatic horses. However, airway neutrophil activation during asthmatic inflammation was otherwise relatively insensitive to GC. The contribution of IL-17 to these responses requires further study.

     

Setting of graded levels of a protein in yeast by a t-degron technique as applied to phosphoglycerate mutase

Background

Setting of graded levels of a protein for in vivo studies by controlled gene expression has inconveniences, and we here explore the use of the t-degron technique instead.

Results

In a yeast t-degron (ubiquitin-argDHFR^ts)- phosphoglycerate mutase (GPM1) fusion strain, increasing periods of exposure to the non-permissive temperature 37°C, even in the presence of cycloheximide, gave decreasing function, as assessed at 23°C in vivo by glucose metabolism and confirmed by immunoblot.

Conclusion

An ideal system would set a range of lower levels of a protein, do so without compensating protein synthesis, and give stable activity for in vitro comparisons. Although the first two aims appear obtainable, the third was not in this example of the application, limiting its uses for some but not all purposes.

     

Effects of specific allergen immunotherapy on biological markers and clinical parameters in asthmatic children: a controlled-real life study

Background

Allergen-specific immunotherapy (AIT) is the only treatment able to change the natural course of allergic diseases. We aimed at investigating the clinical efficacy of SLITOR (Serbian registered vaccine for sublingual allergen specific immunotherapy).

Methods

7–18 years old children with allergic asthma and rhinitis were enrolled and addressed to the active (AIT plus pharmacological treatment) or control (standard pharmacological treatment only) group. Clinical and medications scores, lung function and exhaled FeNO were measured at baseline and at every follow-up.

Results

There was a significant improvement in both nasal and asthma symptom scores as well as in medication score in SLIT group. SLIT showed an important influence on lung function and airway inflammation.

Conclusions

Our data showed that SLITOR was effective not only in terms of patient reported outcomes but an improvement of pulmonary function and decrease of lower airway inflammation were also observed.

     

Nasal gene expression differentiates COPD from controls and overlaps bronchial gene expression

Background

Nasal gene expression profiling is a promising method to characterize COPD non-invasively. We aimed to identify a nasal gene expression profile to distinguish COPD patients from healthy controls. We investigated whether this COPD-associated gene expression profile in nasal epithelium is comparable with the profile observed in bronchial epithelium.

Methods

Genome wide gene expression analysis was performed on nasal epithelial brushes of 31 severe COPD patients and 22 controls, all current smokers, using Affymetrix Human Gene 1.0 ST Arrays. We repeated the gene expression analysis on bronchial epithelial brushes in 2 independent cohorts of mild-to-moderate COPD patients and controls.

Results

In nasal epithelium, 135 genes were significantly differentially expressed between severe COPD patients and controls, 21 being up- and 114 downregulated in COPD (false discovery rate?<?0.01). Gene Set Enrichment Analysis (GSEA) showed significant concordant enrichment of COPD-associated nasal and bronchial gene expression in both independent cohorts (FDR_GSEA <?0.001).

Conclusion

We identified a nasal gene expression profile that differentiates severe COPD patients from controls. Of interest, part of the nasal gene expression changes in COPD mimics differentially expressed genes in the bronchus. These findings indicate that nasal gene expression profiling is potentially useful as a non-invasive biomarker in COPD.

Trial registration

ClinicalTrials.gov registration number NCT01351792 (registration date May 10, 2011), ClinicalTrials.gov registration number NCT00848406 (registration date February 19, 2009), ClinicalTrials.gov registration number NCT00807469 (registration date December 11, 2008).

     

The degradation of nucleotide triphosphates extracted under boiling ethanol conditions is prevented by the yeast cellular matrix

Introduction

Boiling ethanol extraction is a frequently used method for metabolomics studies of biological samples. However, the stability of several central carbon metabolites, including nucleotide triphosphates, and the influence of the cellular matrix on their degradation have not been addressed.

Objectives

To study how a complex cellular matrix extracted from yeast (Saccharomyces cerevisiae) may affect the degradation profiles of nucleotide triphosphates extracted under boiling ethanol conditions.

Methods

We present a double-labelling LC–MS approach with a ¹³C-labeled yeast cellular extract as complex surrogate matrix, and ¹³C¹⁵N-labeled nucleotides as internal standards, to study the effect of the yeast matrix on the degradation of nucleotide triphosphates.

Results

While nucleotide triphosphates were degraded to the corresponding diphosphates in pure solutions, degradation was prevented in the presence of the yeast matrix under typical boiling ethanol extraction conditions.

Conclusions

Extraction of biological samples under boiling ethanol extraction conditions that rapidly inactivate enzyme activity are suitable for labile central energy metabolites such as nucleotide triphosphates due to the stabilizing effect of the yeast matrix. The basis of this phenomenon requires further study.

Graphical abstract

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