The development of a sensitive and specific ELISA for mouse eosinophil peroxidase: Assessment of eosinophil degranulation ex vivo and in models of human disease

Introduction: Clinical significance of tachycardiomyopathy (TCM) increased with trials on catheter ablation therapy. Myocardial biopsies from patients show disturbed mitochondrial architecture. Hypothesis: TCM involves mitochondrial dysfunction. Methods: First, TCM was investigated in an animal model: pacemaker implantation in 7 rabbits was followed by tachypacing for 30 days (TCM), 7 animals served as sham-operated controls (SHAM). Second, results of the animal study were evaluated for their translational perspective for human disease using a novel model of induced pluripotent stem cell-derived cardiomyocytes (iPS-CM), derived from 4 healthy donors. IPS-CM were paced with 120 bpm (TACH) or 60 bpm (CTRL) for 7 days in vitro. Targeted transcriptomics, high-resolution respirometry and flow cytometry (MitoSOX Red) were performed. To account for variations between cell differentiations, experiments on iPS-CM were carried out in a paired design. Results: TCM showed LV dilatation and dysfunction (ΔLVEDD +5.3±0.2mm; ΔFS -19±8%; TCM-SHAM; p<0.001). Histological findings resembled human disease entailing cardiomyocyte hypertrophy (CSA 519±32μm 2 vs. 413±21μm 2 , p<0.01) without fibrosis (hydroxyproline content, p=0.52). Mitochondrial transcriptome of TCM was characterized by downregulation of 10 antioxidative enzymes (e.g. GPX3, fold change (FC) 0.4; TCM/SHAM; p<0.05) as well as mitochondrial carriers, including ADP/ATP- and NADH-shuttling (SLC25A4, FC 0.7; SLC25A12, FC 0.8; p<0.01). As transcriptomics implied impaired substrate import, respirometry was performed in whole tissue. In support of our findings on the transcriptome level, mitochondrial oxidative phosphorylation capacity decreased in TCM (133±13 vs. 170±16 pmol·O 2 ·s -1 ·mg -1 ·tissue, p<0.05). Similarly, oxidative phosphorylation was reduced in iPS-CM (995±738 vs. 1838±901 pmolO 2 ·s -1 ·IU -1 citrate synthase activity, TACH vs. CTRL, p<0.01). Concurrently, tachypacing increased mitochondrial superoxide emission in iPS-CM (MFI 491±206 vs. 301±119, p<0.05). Conclusions: Persistent tachycardia alters two mitochondrial key functions in an animal and a novel human ex vivo model: oxidative phosphorylation capacity is reduced, while superoxide emission increases.

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Gene transfer and delivery in central nervous system disease

Neurosurgical FOCUS ◽

10.3171/foc.1997.3.3.5 ◽

1997 ◽

Vol 3 (3) ◽

pp. E4 ◽

Cited By ~ 1

Author(s):

Gordon Tang ◽

E. Antonio Chiocca

Keyword(s):

Gene Therapy ◽

Central Nervous System ◽

Nervous System ◽

Gene Transfer ◽

Human Disease ◽

Malignant Tumors ◽

Ex Vivo ◽

Central Nervous System Disease ◽

Trophic Factors ◽

Target Tissue

Gene transfer offers the potential to explore basic physiological processes and to intervene in human disease. The central nervous system (CNS) presents a fertile field in which to develop novel therapeutic modalities to treat intractable and pervasive malignant tumors and neurodegenerative disease. The extension of gene therapy to the CNS, however, faces the delivery obstacles of a target population that is postmitotic and isolated behind a blood-brain barrier (BBB). Approaches to this problem have included grafting of genetically modified cells to deliver novel proteins or introducing genes by viral or synthetic vectors geared toward the CNS cell population. Direct inoculation and bulk flow, as well as osmotic and pharmacological disruption, have been used to circumvent the BBB's exclusionary role. Once the gene is delivered, myriad strategies have been used to affect a therapeutic result. Genes activating prodrugs are the most common antitumor approach. Other approaches focus on activating immune responses, targeting angiogenesis, and influencing apoptosis and tumor suppression. At this time, therapy directed at neurodegenerative diseases has centered on ex vivo gene therapy for supply of trophic factors to promote neuronal survival, axonal outgrowth, and target tissue function. Despite early promise, gene therapy for CNS disorders will require advancements in methods for delivery and long-term expression before becoming feasible for human disease.

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Effect of TGF-β1 on eosinophils to induce cysteinyl leukotriene E4 production in aspirin-exacerbated respiratory disease

PLoS ONE ◽

10.1371/journal.pone.0256237 ◽

2021 ◽

Vol 16 (8) ◽

pp. e0256237

Author(s):

Youngwoo Choi ◽

Soyoon Sim ◽

Dong-Hyun Lee ◽

Hee-Ra Lee ◽

Ga-Young Ban ◽

...

Keyword(s):

Respiratory Disease ◽

Transforming Growth Factor Beta ◽

Transforming Growth Factor ◽

Ex Vivo ◽

Cysteinyl Leukotriene ◽

Aspirin Exacerbated Respiratory Disease ◽

Healthy Control ◽

Tgf Β1 ◽

Eosinophil Degranulation

Cysteinyl leukotriene (cysLT) overproduction and eosinophil activation are hallmarks of aspirin-exacerbated respiratory disease (AERD). However, pathogenic mechanisms of AERD remain to be clarified. Here, we aimed to find the significance of transforming growth factor beta 1 (TGF-β1) in association with cysteinyl leukotriene E4 (LTE4) production, leading to eosinophil degranulation. To evaluate levels of serum TGF-β1, first cohort enrolled AERD (n = 336), ATA (n = 442) patients and healthy control subjects (HCs, n = 253). In addition, second cohort recruited AERD (n = 34) and ATA (n = 25) patients to investigate a relation between levels of serum TGF-β1 and urinary LTE4. The function of TGF-β1 in LTE4 production was further demonstrated by ex vivo (human peripheral eosinophils) or in vivo (BALB/c mice) experiment. As a result, the levels of serum TGF-β1 were significantly higher in AERD patients than in ATA patients or HCs (P = .001; respectively). Moreover, levels of serum TGF-β1 and urinary LTE4 had a positive correlation (r = 0.273, P = .037). In the presence of TGF-β1, leukotriene C4 synthase (LTC4S) expression was enhanced in peripheral eosinophils to produce LTE4, which sequentially induced eosinophil degranulation via the p38 pathway. When mice were treated with TGF-β1, significantly induced eosinophilia with increased LTE4 production in the lung tissues were noted. These findings suggest that higher levels of TGF-β1 in AERD patients may contribute to LTE4 production via enhancing LTC4S expression which induces eosinophil degranulation, accelerating airway inflammation.

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Building neuromuscular junctions in vitro

Development ◽

10.1242/dev.193920 ◽

2020 ◽

Vol 147 (22) ◽

pp. dev193920

Author(s):

Susie Barbeau ◽

Julie Tahraoui-Bories ◽

Claire Legay ◽

Cécile Martinat

Keyword(s):

Human Disease ◽

Molecular Mechanisms ◽

Neuromuscular Junctions ◽

Ex Vivo ◽

Culture Methods ◽

Recent Developments ◽

Chemical Synapses ◽

And Function

ABSTRACTThe neuromuscular junction (NMJ) has been the model of choice to understand the principles of communication at chemical synapses. Following groundbreaking experiments carried out over 60 years ago, many studies have focused on the molecular mechanisms underlying the development and physiology of these synapses. This Review summarizes the progress made to date towards obtaining faithful models of NMJs in vitro. We provide a historical approach discussing initial experiments investigating NMJ development and function from Xenopus to mice, the creation of chimeric co-cultures, in vivo approaches and co-culture methods from ex vivo and in vitro derived cells, as well as the most recent developments to generate human NMJs. We discuss the benefits of these techniques and the challenges to be addressed in the future for promoting our understanding of development and human disease.

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A sensitive high throughput ELISA for human eosinophil peroxidase: A specific assay to quantify eosinophil degranulation from patient-derived sources

Journal of Immunological Methods ◽

10.1016/j.jim.2012.06.011 ◽

2012 ◽

Vol 384 (1-2) ◽

pp. 10-20 ◽

Cited By ~ 22

Author(s):

Sergei I. Ochkur ◽

John Dongil Kim ◽

Cheryl A. Protheroe ◽

Dana Colbert ◽

Rachel M. Condjella ◽

...

Keyword(s):

High Throughput ◽

Human Eosinophil ◽

Specific Assay ◽

Eosinophil Peroxidase ◽

Eosinophil Degranulation

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Abstract 309: Blockade of Transforming Growth Factor Beta Activity in Elastase-Induced Aortic Injury in Mice Induces a Human-Like Abdominal Aneurysm

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.37.suppl_1.309 ◽

2017 ◽

Vol 37 (suppl_1) ◽

Author(s):

Marc Clement ◽

Fabien Lareyre ◽

Juliette Raffort ◽

Stefanie Pohlod ◽

Meghana Patel ◽

...

Keyword(s):

Transforming Growth Factor Beta ◽

Human Disease ◽

Transforming Growth Factor ◽

Ex Vivo ◽

Aortic Injury ◽

Aortic Aneurysms ◽

Beta Activity ◽

Aortic Dilatation ◽

Abdominal Aneurysm ◽

Abdominal Aortic

Background: Abdominal aortic aneurysm (AAA) carries important morbidity and mortality and is resistant to medical therapy. Current experimental models do not accurately reproduce the major features of the human disease. There are 2 major categories of mouse models of AAA: those that induce medial dissection, which is not a major characteristic of human AAA, and those that induce aortic dilatation but are self-contained and do not progress to rupture. Methods: We hypothesized that blockade of TGFβ activity, a guardian of vascular integrity and immune homeostasis, and a major causal factor in genetically triggered thoracic aortic aneurysms in humans, would impair vascular healing in models of ‘non-dissecting’ abdominal aortic dilatation, and would lead to continuous aneurysmal growth until rupture. We tested this hypothesis in the elastase-induced abdominal aortic dilatation model in mice. We analyzed AAA development and progression using ultrasound in vivo, advanced synchrotron-based ultrahigh resolution imaging ex-vivo, and a combination of biological, histological and flow cytometry-based cellular and molecular approaches in vitro. Results: We show that systemic blockade of TGFβ activity using a neutralizing mouse monoclonal anti-TGFβ antibody induces a transition from a model of self-contained aortic dilatation to a model of sustained aneurysmal growth culminating in rupture. TGFβ blockade enhances leukocyte infiltration and extracellular matrix degradation, and leads to sustained aneurysmal aortic dilatation, associated with the formation of an intra-luminal thrombus infiltrated with neutrophils, as seen in the human disease. Persistent AAA growth throughout the duration of the experiment is associated with wall disruption without medial dissection, and culminates in fatal aortic wall rupture. Monocyte/macrophage depletion substantially limits AAA severity. Conclusions: Endogenous TGFβ activity is required for the resolution of elastase-induced aortic injury. We expect that this new model will improve our understanding of the pathophysiology of AAA, and will be useful to identify new therapeutic targets.

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Use of Protein a Conjugated to Peroxidase to Localize Immunoglobulin G in SSPE Ferret Brain

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100054376 ◽

1982 ◽

Vol 40 ◽

pp. 350-351

Author(s):

Hannah R. Brown ◽

Anthony F. Nostro ◽

Halldor Thormar

Keyword(s):

Immunoglobulin G ◽

Human Disease ◽

Measles Virus ◽

Subacute Sclerosing Panencephalitis ◽

Protein A ◽

Inflammatory Lesions ◽

Sspe Virus ◽

Specific Immunoglobulin ◽

Antibody Titers ◽

The Brain

Subacute sclerosing panencephalitis (SSPE) is a slowly progressing disease of the CNS in children which is caused by measles virus. Ferrets immunized with measles virus prior to inoculation with the cell associated, syncytiogenic D.R. strain of SSPE virus exhibit characteristics very similar to the human disease. Measles virus nucleocapsids are present, high measles antibody titers are found in the sera and inflammatory lesions are prominent in the brains. Measles virus specific immunoglobulin G (IgG) is present in the brain,and IgG/ albumin ratios indicate that the antibodies are synthesized within the CNS.

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Spectroscopic imaging of human disease

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100166889 ◽

1996 ◽

Vol 54 ◽

pp. 884-885

Author(s):

D.J. Meyerhoff

Keyword(s):

Magnetic Field ◽

Magnetic Resonance ◽

Field Gradient ◽

Human Disease ◽

Morphological Changes ◽

Magnetic Field Gradient ◽

Spectroscopic Imaging ◽

Resonance Spectroscopy ◽

Tissue Water

Magnetic Resonance Imaging (MRI) observes tissue water in the presence of a magnetic field gradient to study morphological changes such as tissue volume loss and signal hyperintensities in human disease. These changes are mostly non-specific and do not appear to be correlated with the range of severity of a certain disease. In contrast, Magnetic Resonance Spectroscopy (MRS), which measures many different chemicals and tissue metabolites in the millimolar concentration range in the absence of a magnetic field gradient, has been shown to reveal characteristic metabolite patterns which are often correlated with the severity of a disease. In-vivo MRS studies are performed on widely available MRI scanners without any “sample preparation” or invasive procedures and are therefore widely used in clinical research. Hydrogen (H) MRS and MR Spectroscopic Imaging (MRSI, conceptionally a combination of MRI and MRS) measure N-acetylaspartate (a putative marker of neurons), creatine-containing metabolites (involved in energy processes in the cell), choline-containing metabolites (involved in membrane metabolism and, possibly, inflammatory processes),

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