scholarly journals The neutrophil-mobilizing cytokine interleukin-26 in the airways of long-term tobacco smokers

2018 ◽  
Vol 132 (9) ◽  
pp. 959-983 ◽  
Author(s):  
Karlhans Fru Che ◽  
Ellen Tufvesson ◽  
Sara Tengvall ◽  
Elisa Lappi-Blanco ◽  
Riitta Kaarteenaho ◽  
...  
Keyword(s):  
Gene Expression ◽  
Chronic Bronchitis ◽  
Pathogenic Bacteria ◽  
Cell Model ◽  
Water Soluble ◽  
Chronic Obstructive ◽  
Neutrophil Accumulation

Long-term tobacco smokers with chronic obstructive pulmonary disease (COPD) or chronic bronchitis display an excessive accumulation of neutrophils in the airways; an inflammation that responds poorly to established therapy. Thus, there is a need to identify new molecular targets for the development of effective therapy. Here, we hypothesized that the neutrophil-mobilizing cytokine interleukin (IL)-26 (IL-26) is involved in airway inflammation amongst long-term tobacco smokers with or without COPD, chronic bronchitis or colonization by pathogenic bacteria. By analyzing bronchoalveolar lavage (BAL), bronchail wash (BW) and induced sputum (IS) samples, we found increased extracellular IL-26 protein in the airways of long-term smokers in vivo without further increase amongst those with clinically stable COPD. In human alveolar macrophages (AM) in vitro, the exposure to water-soluble tobacco smoke components (WTC) enhanced IL-26 gene and protein. In this cell model, the same exposure increased gene expression of the IL-26 receptor complex (IL10R2 and IL20R1) and nuclear factor κ B (NF-κB); a proven regulator of IL-26 production. In the same cell model, recombinant human IL-26 in vitro caused a concentration-dependent increase in the gene expression of NF-κB and several pro-inflammatory cytokines. In the long-term smokers, we also observed that extracellular IL-26 protein in BAL samples correlates with measures of lung function, tobacco load, and several markers of neutrophil accumulation. Extracellular IL-26 was further increased in long-term smokers with exacerbations of COPD (IS samples), with chronic bronchitis (BAL samples ) or with colonization by pathogenic bacteria (IS and BW samples). Thus, IL-26 in the airways emerges as a promising target for improving the understanding of the pathogenic mechanisms behind several pulmonary morbidities in long-term tobacco smokers.

scholarly journals c-Jun N-Terminal Kinase Activation of Activator Protein-1 Underlies Homologous Regulation of the Gonadotropin-Releasing Hormone Receptor Gene in αT3-1 Cells

Endocrinology ◽  
2003 ◽  
Vol 144 (3) ◽  
pp. 839-849 ◽  
Author(s):  
Buffy S. Ellsworth ◽  
Brett R. White ◽  
Ann T. Burns ◽  
Brian D. Cherrington ◽  
Annette M. Otis ◽  
...  
Keyword(s):  
Gene Expression ◽  
Protein Kinase ◽  
Cell Model ◽  
Receptor Gene ◽  
Critical Role ◽  
Dominant Negative ◽  
Activator Protein 1 ◽  
Activator Protein

Reproductive function is dependent on the interaction between GnRH and its cognate receptor found on gonadotrope cells of the anterior pituitary gland. GnRH activation of the GnRH receptor (GnRHR) is a potent stimulus for increased expression of multiple genes including the gene encoding the GnRHR itself. Thus, homologous regulation of the GnRHR is an important mechanism underlying gonadotrope sensitivity to GnRH. Previously, we have found that GnRH induction of GnRHR gene expression in αT3-1 cells is partially mediated by protein kinase C activation of a canonical activator protein-1 (AP-1) element. In contrast, protein kinase A and a cAMP response element-like element have been implicated in mediating the GnRH response of the GnRHR gene using a heterologous cell model (GGH3). Herein we find that selective removal of the canonical AP-1 site leads to a loss of GnRH regulation of the GnRHR promoter in transgenic mice. Thus, an intact AP-1 element is necessary for GnRH responsiveness of the GnRHR gene both in vitro and in vivo. Based on in vitro analyses, GnRH appeared to enhance the interaction of JunD, FosB, and c-Fos at the GnRHR AP-1 element. Although enhanced binding of cFos reflected an increase in gene expression, GnRH appeared to regulate both FosB and JunD at a posttranslational level. Neither overexpression of a constitutively active Raf-kinase nor pharmacological blockade of GnRH-induced ERK activation eliminated the GnRH response of the GnRHR promoter. GnRH responsiveness was, however, lost in αT3-1 cells that stably express a dominant-negative c-Jun N-terminal kinase (JNK) kinase, suggesting a critical role for JNK in mediating GnRH regulation of the GnRHR gene. Consistent with this possibility, we find that the ability of forskolin and membrane-permeable forms of cAMP to inhibit the GnRH response of the GnRHR promoter is associated with a loss of both JNK activation and GnRH-mediated recruitment of the primary AP-1-binding components.

scholarly journals Long-term evolution of antibiotic persistence in P. aeruginosa lung infections

2021 ◽  
Author(s):  
Melanie Ghoul ◽  
Sandra B Andersen ◽  
Helle Krogh Johansen ◽  
Lars Jelsbak ◽  
Søren Molin ◽  
...  
Keyword(s):  
Pathogenic Bacteria ◽  
Resistance Mechanisms ◽  
Term Evolution ◽  
Bacterial Clone ◽  
Evolution Of Resistance ◽  
Lung Infections

Pathogenic bacteria respond to antibiotic pressure with the evolution of resistance but survival can also depend on their ability to tolerate antibiotic treatment, known as persistence. While a variety of resistance mechanisms and underlying genetics are well characterised in vitro and in vivo, the evolution of persistence, and how it interacts with resistance in situ is less well understood. We assayed for persistence and resistance with three clinically relevant antibiotics: meropenem, ciprofloxacin and tobramycin, in isolates of Pseudomonas aeruginosa from chronic cystic fibrosis lung infections spanning up to forty years of evolution. We find evidence that persistence is under positive selection in the lung and that it can particularly act as an evolutionary stepping stone to resistance. However, this pattern is not universal and depends on the bacterial clone type and antibiotic used, indicating an important role for antibiotic mode of action.

scholarly journals Genipin Reverses HFD-Induced Liver Damage and Inhibits UCP2-Mediated Pyroptosis in Mice

2018 ◽  
Vol 49 (5) ◽  
pp. 1885-1897 ◽  
Author(s):  
Hong Zhong ◽  
Mengting Liu ◽  
Yaya Ji ◽  
Minjuan Ma ◽  
Kun Chen ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Death ◽  
Lactate Dehydrogenase ◽  
Liver Damage ◽  
Lipid Accumulation ◽  
Water Soluble ◽  
Morbidly Obese ◽  
Ldh Release

Background/Aims: Liver damage is a typical manifestation of nonalcoholic fatty liver disease (NAFLD). It originates from excessive fat accumulation, leading to hepatocyte death, inflammation, and fibrosis. Nonalcoholic steatohepatitis (NASH) is a type of NAFLD with a prevalence of 49% in morbidly obese patients. Pyroptosis plays an important role in the development of NASH; thus, it is important to elucidate the effect of lipid accumulation on pyroptosis. Genipin (GNP), a natural water-soluble cross-linking agent, has hepatoprotective effects and decreases lipid accumulation in the liver; however, the mechanisms underlying these effects are unknown. Methods: In this study, qPCR and Western blot were used to examine pyroptotic gene expression in high-fat diet (HFD) induced obese mice and free fatty acids (FFAs) treated hepatocytes. At the same time, relative lactate dehydrogenase (LDH) release and Hoechst & propidium iodide (PI) staining were done to verify cell death. To explore the molecular mechanism, cell transfection were constructed with siRNA or plasmid to obtain knockdown or overexpression hepatocytes. Results: We found that HFD-fed mice and FFAs-treated hepatocytes had obvious pyroptosis, and addition of GNP reversed liver damage and inhibited pyroptosis both in vitro and in vivo. Besides, UCP2 knockdown cells showed suppressed FFAs-mediated pyroptosis, as determined by decreased pyroptotic gene expression, reduced lactate dehydrogenase (LDH) release, and reduced cell death. Consistent with this, cells transfected with UCP2 had upregulated pyroptotic gene expression, increased LDH release, and increased cell death. Conclusion: GNP reverses HFD-induced liver damage and inhibits UCP2-mediated pyroptosis. Thus, GNP may serve as a potential therapeutic candidate for NAFLD.

scholarly journals Novel Transcriptional Regulatory Signals in the Adeno-Associated Virus Terminal Repeat A/D Junction Element

2000 ◽  
Vol 74 (18) ◽  
pp. 8732-8739 ◽  
Author(s):  
Rebecca P. Haberman ◽  
Thomas J. McCown ◽  
Richard Jude Samulski
Keyword(s):  
Gene Expression ◽  
Fluorescent Protein ◽  
Terminal Repeat ◽  
Cmv Promoter ◽  
Adeno Associated Virus ◽  
Regulated Expression ◽  
Regulated Gene Expression

ABSTRACT Adeno-associated virus (AAV) type 2 vectors transfer stable, long-term gene expression to diverse cell types in vivo. Many gene therapy applications require the control of long-term transgene expression, and AAV vectors, similar to other gene transfer systems, are being evaluated for delivery of regulated gene expression cassettes. Previously, we (R. P. Haberman, T. J. McCown, and R. J. Samulski, Gene Ther. 5:1604–1611, 1998) demonstrated the use of the tetracycline-responsive system for long-term regulated expression in rat brains. In that study, we also observed residual expression in the “off” state both in vitro and in vivo, suggesting that the human cytomegalovirus (CMV) major immediate-early minimal promoter or other cis-acting elements (AAV terminal repeats [TR]) were contributing to this activity. In the present study, we identify that the AAV TR, minus the tetracycline-responsive minimal CMV promoter, will initiate mRNA expression from vector templates. Using deletion analysis and specific PCR-derived TR reporter gene templates, we mapped this activity to a 37-nucleotide stretch in theA/D elements of the TR. Although the mRNA derived from the TR is generated from a non-TATA box element, the use of mutant templates failed to identify function of canonical initiator sequences as previously described. Finally, we demonstrated the presence of green fluorescent protein expression both in vitro and in vivo in brain by using recombinant virus carrying only the TR element. Since the AAV terminal repeat is a necessary component of all recombinant AAV vectors, this TR transcriptional activity may interfere with all regulated expression cassettes and may be a problem in the development of novel TR split gene vectors currently being considered for genes too large to be packaged.

scholarly journals Tocotrienols: Dietary Supplements for Chronic Obstructive Pulmonary Disease

Antioxidants ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 883
Author(s):  
Xiangming Ji ◽  
Hongwei Yao ◽  
Maureen Meister ◽  
Douglas S. Gardenhire ◽  
Huanbiao Mo
Keyword(s):  
Chronic Obstructive Pulmonary Disease ◽  
Chronic Bronchitis ◽  
Pulmonary Disease ◽  
Significant Risk ◽  
Chronic Obstructive ◽  
Alveolar Wall ◽  
Obstructive Pulmonary Disease ◽  
Anti Inflammatory

Chronic obstructive pulmonary disease (COPD) is one of the leading causes of death worldwide. Emphysema and chronic bronchitis are the two major phenotypes of COPD, which have many symptoms, such as dyspnea, chronic cough, and mucus overproduction. Emphysema is characterized by the destruction of the alveolar wall, while chronic bronchitis is characterized by limitations in expiratory airflow. Cigarette smoking is the most significant risk factor for the pathogenesis of COPD in the developed world. Chronic inflammation contributes to the onset and progression of the disease and furthers the risk of comorbidities. Current treatment options and prevention strategies for COPD are very limited. Tocotrienols are a group of vitamin E molecules with antioxidant and anti-inflammatory properties. Individual tocotrienols (α, γ, and δ) have shown their ability to attenuate inflammation specifically via suppressing nuclear factor-κB-mediated cytokine production. The δ- and γ-forms of tocotrienols have been indicated as the most effective in the prevention of macrophage infiltration, production of reactive oxygen species, and cytokine secretion. This review briefly discusses the pathogenesis of COPD and the role of inflammation therein. Furthermore, we summarize the in vitro and in vivo evidence for the anti-inflammatory activity of tocotrienols and their potential application to COPD management. Coupled with the bioavailability and safety profile of tocotrienols, the ability of these compounds to modulate COPD progression by targeting the inflammation pathways renders them potential candidates for novel therapeutic approaches in the treatment of COPD patients.

scholarly journals 3D Bioprinting Allows the Establishment of Long-Term 3D Culture Model for Chronic Lymphocytic Leukemia Cells

2021 ◽  
Vol 12 ◽  
Author(s):  
Francesca Vittoria Sbrana ◽  
Riccardo Pinos ◽  
Federica Barbaglio ◽  
Davide Ribezzi ◽  
Fiorella Scagnoli ◽  
...  
Keyword(s):  
Gene Expression ◽  
3D Culture ◽  
Lymphocytic Leukemia ◽  
Leukemic Cells ◽  
3D Bioprinting ◽  
Culture Model ◽  
3D Culture Model

Chronic Lymphocytic Leukemia (CLL) represents the most common leukemia in the western world and remains incurable. Leukemic cells organize and interact in the lymphoid tissues, however what actually occurs in these sites has not been fully elucidated yet. Studying primary CLL cells in vitro is very challenging due to their short survival in culture and also to the fact that traditional two-dimensional in vitro models lack cellular and spatial complexity present in vivo. Based on these considerations, we exploited for the first time three-dimensional (3D) bioprinting to advance in vitro models for CLL. This technology allowed us to print CLL cells (both primary cells and cell lines) mixed with the appropriate, deeply characterized, hydrogel to generate a scaffold containing the cells, thus avoiding the direct cell seeding onto a precast 3D scaffold and paving the way to more complex models. Using this system, we were able to efficiently 3D bioprint leukemic cells and improve their viability in vitro that could be maintained up to 28 days. We monitored over time CLL cells viability, phenotype and gene expression, thus establishing a reproducible long-term 3D culture model for leukemia. Through RNA sequencing (RNAseq) analysis, we observed a consistent difference in gene expression profile between 2D and 3D samples, indicating a different behavior of the cells in the two different culture settings. In particular, we identified pathways upregulated in 3D, at both day 7 and 14, associated with immunoglobulins production, pro-inflammatory molecules expression, activation of cytokines/chemokines and cell-cell adhesion pathways, paralleled by a decreased production of proteins involved in DNA replication and cell division, suggesting a strong adaptation of the cells in the 3D culture. Thanks to this innovative approach, we developed a new tool that may help to better mimic the physiological 3D in vivo settings of leukemic cells as well as of immune cells in broader terms. This will allow for a more reliable study of the molecular and cellular interactions occurring in normal and neoplastic conditions in vivo, and could also be exploited for clinical purposes to test individual responses to different drugs.

scholarly journals Analysis of gene transcription alterations at the blastocyst stage related to the long-term consequences of in vitro culture in mice

Reproduction ◽  
2009 ◽  
Vol 137 (2) ◽  
pp. 271-283 ◽  
Author(s):  
Raúl Fernández-González ◽  
Juan de Dios Hourcade ◽  
Irene López-Vidriero ◽  
Alberto Benguría ◽  
Fernando Rodríguez De Fonseca ◽  
...  
Keyword(s):  
Gene Expression ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Blastocyst Stage ◽  
Epigenetic Mechanisms ◽  
Long Term Effects ◽  
Long Term Consequences

We have reported thatin vitroculture (IVC) of preimplantation mouse embryos in the presence of FCS produces long-term effects (LTE) on development, growth and behaviour of the offspring at adult age. To analyse the mechanisms underlying this phenomenon, we have examined development and global alterations in gene expression in the mouse blastocysts produced in the presence of FCS, conditions known to be suboptimal and that generate LTE. Embryos culturedin vitroin KSOM and in KSOM+FCS had a reduced number of cells in the inner cell mass at the blastocyst stage compared within vivoderived embryos; however, only culture in KSOM+FCS leads to a reduction in the number of trophoblast cells. Gene expression levels were measured by comparison among three groups of blastocysts (in vivo, IVC in KSOM and IVC in KSOM+FCS). Different patterns of gene expression and development were found between embryos culturedin vitroorin vivo. Moreover, when we compared the embryos produced in KSOM versus KSOM+FCS, we observed that the presence of FCS affected the expression of 198 genes. Metabolism, proliferation, apoptosis and morphogenetic pathways were the most common processes affected by IVC. However, the presence of FCS during IVC preferentially affected genes associated with certain molecular and biological functions related to epigenetic mechanisms. These results suggest that culture-induced alterations in transcription at the blastocyst stage related to epigenetic mechanisms provide a foundation for understanding the molecular origin at the time of preimplantation development of the long-term consequences of IVC in mammals.

scholarly journals Toxicity and Functional Impairment in Human Adipose Tissue-Derived Stromal Cells (hASCs) Following Long-Term Exposure to Very Small Iron Oxide Particles (VSOPs)

Nanomaterials ◽  
2020 ◽  
Vol 10 (4) ◽  
pp. 741 ◽  
Author(s):  
Katrin Radeloff ◽  
Andreas Radeloff ◽  
Mario Ramos Tirado ◽  
Agmal Scherzad ◽  
Rudolf Hagen ◽  
...  
Keyword(s):  
Gene Expression ◽  
Adipose Tissue ◽  
Iron Oxide ◽  
Stromal Cells ◽  
Caspase 3 ◽  
Human Adipose Tissue ◽  
Labelling Procedure

Magnetic nanoparticles (NPs), such as very small iron oxide NPs (VSOPs) can be used for targeted drug delivery, cancer treatment or tissue engineering. Another important field of application is the labelling of mesenchymal stem cells to allow in vivo tracking and visualization of transplanted cells using magnetic resonance imaging (MRI). For these NPs, however, various toxic effects, as well as functional impairment of the exposed cells, are described. The present study evaluates the influence of VSOPs on the multilineage differentiation ability and cytokine secretion of human adipose tissue derived stromal cells (hASCs) after long-term exposure. Human ASCs were labelled with VSOPs, and the efficacy of the labelling was documented over 4 weeks in vitro cultivation of the labelled cells. Unlabelled hASCs served as negative controls. Four weeks after labelling, adipogenic and osteogenic differentiation was histologically evaluated and quantified by polymerase chain reaction (PCR). Changes in gene expression of IL-6, IL-8, VEGF and caspase 3 were determined over 4 weeks. Four weeks after the labelling procedure, labelled and unlabelled hASCs did not differ in the gene expression of IL-6, IL-8, VEGF and caspase 3. Furthermore, the labelling procedure had no influence on the multidifferentiation ability of hASC. The percentage of labelled cells decreased during in vitro expansion over 4 weeks. Labelling with VSOPs and long-term intracellular disposition probably have no influence on the physiological functions of hASCs. This could be important for the future in vivo use of iron oxide NPs.

Endogenous hydrogen sulfide sulfhydrates IKKβ at cysteine 179 to control pulmonary artery endothelial cell inflammation

2019 ◽  
Vol 133 (20) ◽  
pp. 2045-2059 ◽  
Author(s):  
Da Zhang ◽  
Xiuli Wang ◽  
Siyao Chen ◽  
Selena Chen ◽  
Wen Yu ◽  
...  
Keyword(s):  
Hydrogen Sulfide ◽  
Endothelial Cell ◽  
Pulmonary Artery ◽  
Cell Model ◽  
Site Directed Mutagenesis ◽  
Critical Event ◽  
Hypertensive Rats ◽  
Pulmonary Artery Endothelial Cell

Abstract Background: Pulmonary artery endothelial cell (PAEC) inflammation is a critical event in the development of pulmonary arterial hypertension (PAH). However, the pathogenesis of PAEC inflammation remains unclear. Methods: Purified recombinant human inhibitor of κB kinase subunit β (IKKβ) protein, human PAECs and monocrotaline-induced pulmonary hypertensive rats were employed in the study. Site-directed mutagenesis, gene knockdown or overexpression were conducted to manipulate the expression or activity of a target protein. Results: We showed that hydrogen sulfide (H2S) inhibited IKKβ activation in the cell model of human PAEC inflammation induced by monocrotaline pyrrole-stimulation or knockdown of cystathionine γ-lyase (CSE), an H2S generating enzyme. Mechanistically, H2S was proved to inhibit IKKβ activity directly via sulfhydrating IKKβ at cysteinyl residue 179 (C179) in purified recombinant IKKβ protein in vitro, whereas thiol reductant dithiothreitol (DTT) reversed H2S-induced IKKβ inactivation. Furthermore, to demonstrate the significance of IKKβ sulfhydration by H2S in the development of PAEC inflammation, we mutated C179 to serine (C179S) in IKKβ. In purified IKKβ protein, C179S mutation of IKKβ abolished H2S-induced IKKβ sulfhydration and the subsequent IKKβ inactivation. In human PAECs, C179S mutation of IKKβ blocked H2S-inhibited IKKβ activation and PAEC inflammatory response. In pulmonary hypertensive rats, C179S mutation of IKKβ abolished the inhibitory effect of H2S on IKKβ activation and pulmonary vascular inflammation and remodeling. Conclusion: Collectively, our in vivo and in vitro findings demonstrated, for the first time, that endogenous H2S directly inactivated IKKβ via sulfhydrating IKKβ at Cys179 to inhibit nuclear factor-κB (NF-κB) pathway activation and thereby control PAEC inflammation in PAH.

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