New in situ mouse model to quantify alveolar epithelial fluid clearance

1998 ◽  
Vol 84 (5) ◽  
pp. 1763-1767 ◽  
Author(s):  
Chrystelle Garat ◽  
Ethan P. Carter ◽  
Michael A. Matthay
Keyword(s):  
Mouse Model ◽  
Ex Vivo ◽  
Progressive Increase ◽  
Adrenergic Stimulation ◽  
Alveolar Epithelial ◽  
Rat Lungs ◽  
Mouse Lungs ◽  
Experimental Preparation

Because the availability of transgenic mice makes it possible to examine the contribution of single genes to in vivo function, we developed a simple in situ mouse model that can be used to quantify isosmolar alveolar epithelial fluid clearance (AFC). Mice were killed, a tracheostomy was done, and then a test solution of a 5% isosmolar albumin solution with 0.1 μCi of125I-labeled albumin was instilled via the trachea into the distal air spaces of both lungs. After instillation, the lungs were inflated to 7 cmH2O with 100% O2 and maintained at 37°C by placing the animals under an infrared lamp. AFC was measured by the progressive increase in concentration of labeled and unlabeled protein over 1 h. The results indicated the following. 1) Basal, unstimulated AFC in mouse lungs was significantly faster than in ex vivo rat lungs (27 ± 5% in in situ mice vs. 11 ± 3% in ex vivo rat lungs; P < 0.05). 2) Comparison of equivalent doses (10−4 M) of β-adrenergic agonist (isoproterenol) and β2-adrenergic agonists (terbutaline and salmeterol) indicated that stimulated clearance occurred only in presence of isoproterenol. 3) Because atenolol, a specific β1-antagonist, abolished the effect of isoproterenol, the β-adrenergic stimulation appears to be mediated by β1-receptors. The rate of AFC in nonperfused mouse lungs was significantly faster than in prior studies of nonperfused lungs in rats and sheep. Interestingly, the stimulated clearance rate in mice was similar to the fast rates of AFC that we recently reported in patients recovering from hydrostatic pulmonary edema. This in situ model is a unique experimental preparation that can be readily used to quantify isosmolar epithelial fluid clearance in mice.

scholarly journals TMOD-24. GENERATION AND CHARACTERIZATION OF A NOVEL TRANSGENIC IDO REPORTER MOUSE FOR IDO POSTTRANSLATIONAL MODIFICATION ANALYSIS IN SITU

2020 ◽  
Vol 22 (Supplement_2) ◽  
pp. ii233-ii233
Author(s):  
April Bell ◽  
Lijie Zhai ◽  
Erik Ladomersky ◽  
Kristen Lauing ◽  
Lakshmi Bollu ◽  
...  
Keyword(s):  
Mouse Model ◽  
Tumor Cell ◽  
Central Nervous System Tumor ◽  
Post Translational Modifications ◽  
Reporter Mouse ◽  
C Terminus ◽  
Human Gbm

Abstract Glioblastoma (GBM) is the most common and aggressive primary central nervous system tumor in adults with a median survival of 14.6 months. GBM is a potently immunosuppressive cancer due in-part to the prolific expression of immunosuppressive indoleamine 2,3 dioxygenase 1 (IDO). Tumor cell IDO facilitates the intratumoral accumulation of regulatory T cells (Tregs; CD4+CD25+FoxP3+). Although immunosuppressive IDO activity is canonically characterized by the conversion of tryptophan into kynurenine, we have utilized transgenic and syngeneic mouse models and mutant glioma lines to demonstrate that tumor cell IDO increases Treg accumulation independent of tryptophan metabolism. Here, we address the gap in our understanding of IDO signaling activity in vivo. Subcutaneously-engrafted human GBM expressing human IDO-GFP cDNA was isolated from immunodeficient humanized NSG-SGM3 mice. The tumor was immunoprecipitated for the GFP tag using GFP-TRAP followed by mass spectrometry which revealed a novel methylation site on a lysine residue at amino acid 373 in the IDO C-terminus region. Western blot analysis of IDO protein also revealed the presence of tyrosine phosphorylation. Additionally, we recently created a new transgenic IDO reporter mouse model whereby endogenous IDO is fused to GFP via a T2A linker (IDO→GFP). This model allows for the isolation of IDO+ cells in real-time and without causing cell death, thereby creating the opportunity for downstream molecular analysis of in situ-isolated GFP+ cells. Collectively, our work suggests that IDO non-enzyme activity may involve the post-translational modifications we recently identified. As IDO activity may differ between in vitro and in vivo modeling systems, we will use the new IDO→GFP reporter mouse model for an improved mechanistic understanding of how immunosuppressive IDO facilitates Treg accumulation in vivo.

A novel mouse model based on intersectional genetics enables unambiguous in vivo discrimination between plasmacytoid and other dendritic cells and their comparative characterization

2022 ◽  
Author(s):  
Michael Valente ◽  
Nils Collinet ◽  
Thien-Phong Vu Manh ◽  
Karima Naciri ◽  
Gilles Bessou ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
Steady State ◽  
Mouse Model ◽  
Single Cell ◽  
Ex Vivo ◽  
High Specificity ◽  
Type I ◽  
Specific Expression ◽  
Physiological Functions

Plasmacytoid dendritic cells (pDC) were identified about 20 years ago, based on their unique ability to rapidly produce copious amounts of all subsets of type I and type III interferon (IFN-I/III) upon virus sensing, while being refractory to infection. Yet, the identity and physiological functions of pDC are still a matter of debate, in a large part due to their lack of specific expression of any single cell surface marker or gene that would allow to track them in tissues and to target them in vivo with high specificity and penetrance. Indeed, recent studies showed that previous methods that were used to identify or deplete pDC also targeted other cell types, including pDC-like cells and transitional DC (tDC) that were proposed to be responsible for all the antigen presentation ability previously attributed to steady state pDC. Hence, improving our understanding of the nature and in vivo choreography of pDC physiological functions requires the development of novel tools to unambiguously identify and track these cells, including in comparison to pDC-like cells and tDC. Here, we report successful generation of a pDC-reporter mouse model, by using an intersectional genetic strategy based on the unique co-expression of Siglech and Pacsin1 in pDC. This pDC-Tomato mouse strain allows specific ex vivo and in situ detection of pDC. Breeding them with Zbtb46GFP mice allowed side-by-side purification and transcriptional profiling by single cell RNA sequencing of bona fide pDC, pDC-like cells and tDC, in comparison to type 1 and 2 conventional DC (cDC1 and cDC2), both at steady state and during a viral infection, revealing diverging activation patterns of pDC-like cells and tDC. Finally, by breeding pDC-Tomato mice with Ifnb1EYFP mice, we determined the choreography of pDC recruitment to the micro-anatomical sites of viral replication in the spleen, with initially similar but later divergent behaviors of the pDC that engaged or not into IFN-I production. Our novel pDC-Tomato mouse model, and newly identified gene modules specific to combinations of DC types and activations states, will constitute valuable resources for a deeper understanding of the functional division of labor between DC types and its molecular regulation at homeostasis and during viral infections.

In-vitro, ex-vivo, and in-vivo release evaluation of in situ forming buprenorphine implants using mixture of PLGA copolymers and additives

2018 ◽  
Vol 68 (16) ◽  
pp. 965-977 ◽  
Author(s):  
Hossein Kamali ◽  
Elham Khodaverdi ◽  
Farzin Hadizadeh ◽  
Seyed Ahmad Mohajeri ◽  
Younes Kamali ◽  
...  
Keyword(s):  
Ex Vivo ◽  
In Situ Forming ◽  
In Vivo Release

Antibodies to β-Amyloid Decrease the Blood-to-Brain Transfer of β-Amyloid Peptide1

2002 ◽  
Vol 227 (8) ◽  
pp. 609-615 ◽  
Author(s):  
Weihong Pan ◽  
Beka Solomon ◽  
Lawrence M. Maness ◽  
Abba J. Kastin
Keyword(s):  
Ex Vivo ◽  
Amyloid Β ◽  
Brain Perfusion ◽  
Brain Parenchyma ◽  
Amyloid Angiopathy ◽  
Β Amyloid ◽  
Blocking Antibodies ◽  
The Brain

Amyloid-β peptides (Aβ) play an important role in the pathophysiology of dementia of the Alzheimer's type and in amyloid angiopathy. Aβ outside the CNS could contribute to plaque formation in the brain where its entry would involve interactions with the blood-brain barrier (BBB). Effective antibodies to Aβ have been developed in an effort to vaccinate against Alzheimer's disease. These antibodies could interact with Aβ in the peripheral blood, block the passage of Aβ across the BBB, or prevent Aβ deposition within the CNS. To determine whether the blocking antibodies act at the BBB level, we examined the influx of radiolabeled Aβ (125I-Aβ1-40) into the brain after ex-vivo incubation with the antibodies. Antibody mAb3D6 (élan Company) reduced the blood-to-brain influx of Aβ after iv bolus injection. It also significantly decreased the accumulation of Aβ in brain parenchyma. To confirm the in-vivo study and examine the specificity of mAb3D6, in-situ brain perfusion in serum-free buffer was performed after incubation of 125I-Aβ1-40 with another antibody mAbmc1 (DAKO Company). The presence of mAbmc1 also caused significant reduction of the influx of Aβ into the brain after perfusion. Therefore, effective antibodies to Aβ can reduce the influx of Aβ1-40 into the brain.

Apoptosis and DNA damage in type 2 alveolar epithelial cells cultured from hyperoxic rats

1998 ◽  
Vol 274 (5) ◽  
pp. L714-L720 ◽  
Author(s):  
Sue Buckley ◽  
Lora Barsky ◽  
Barbara Driscoll ◽  
Kenneth Weinberg ◽  
Kathryn D. Anderson ◽  
...  
Keyword(s):  
Dna Damage ◽  
Epithelial Cells ◽  
Cellular Response ◽  
Ex Vivo ◽  
Keratinocyte Growth Factor ◽  
Alveolar Epithelial Cells ◽  
Alveolar Epithelial ◽  
Dna Laddering

Apoptosis is a genetically controlled cellular response to developmental stimuli and environmental insult that culminates in cell death. Sublethal hyperoxic injury in rodents is characterized by a complex but reproducible pattern of lung injury and repair during which the alveolar surface is damaged, denuded, and finally repopulated by type 2 alveolar epithelial cells (AEC2). Postulating that apoptosis might occur in AEC2 after hyperoxic injury, we looked for the hallmarks of apoptosis in AEC2 from hyperoxic rats. A pattern of increased DNA end labeling, DNA laddering, and induction of p53, p21, and Bax proteins, strongly suggestive of apoptosis, was seen in AEC2 cultured from hyperoxic rats when compared with control AEC2. In contrast, significant apoptosis was not detected in freshly isolated AEC2 from oxygen-treated rats. Thus the basal culture conditions appeared to be insufficient to ensure the ex vivo survival of AEC2 damaged in vivo. The oxygen-induced DNA strand breaks were blocked by the addition of 20 ng/ml of keratinocyte growth factor (KGF) to the culture medium from the time of plating and were partly inhibited by Matrigel or a soluble extract of Matrigel. KGF treatment resulted in a partial reduction in the expression of the p21, p53, and Bax proteins but had no effect on DNA laddering. We conclude that sublethal doses of oxygen in vivo cause damage to AEC2, resulting in apoptosis in ex vivo culture, and that KGF can reduce the oxygen-induced DNA damage. We speculate that KGF plays a role as a survival factor in AEC2 by limiting apoptosis in the lung after acute hyperoxic injury.

scholarly journals The CD8+ Dendritic Cell Subset Selectively Endocytoses Dying Cells in Culture and In Vivo

2002 ◽  
Vol 195 (10) ◽  
pp. 1289-1302 ◽  
Author(s):  
Tomonori Iyoda ◽  
Susumu Shimoyama ◽  
Kang Liu ◽  
Yoshiki Omatsu ◽  
Yuji Akiyama ◽  
...  
Keyword(s):  
T Cells ◽  
Cd8 T Cells ◽  
Ex Vivo ◽  
Osmotic Shock ◽  
Cell Subset ◽  
Fluorescent Labeling ◽  
Dendritic Cell Subset ◽  
Dying Cells

Dendritic cells (DCs) are able in tissue culture to phagocytose and present antigens derived from infected, malignant, and allogeneic cells. Here we show directly that DCs in situ take up these types of cells after fluorescent labeling with carboxyfluorescein succinimidyl ester (CFSE) and injection into mice. The injected cells include syngeneic splenocytes and tumor cell lines, induced to undergo apoptosis ex vivo by exposure to osmotic shock, and allogeneic B cells killed by NK cells in situ. The CFSE-labeled cells in each case are actively endocytosed by DCs in vivo, but only the CD8+ subset. After uptake, all of the phagocytic CD8+ DCs can form major histocompatibility complex class II–peptide complexes, as detected with a monoclonal antibody specific for these complexes. The CD8+ DCs also selectively present cell-associated antigens to both CD4+ and CD8+ T cells. Similar events take place with cultured DCs; CD8+ DCs again selectively take up and present dying cells. In contrast, both CD8+ and CD8− DCs phagocytose latex particles in culture, and both DC subsets present soluble ovalbumin captured in vivo. Therefore CD8+ DCs are specialized to capture dying cells, and this helps to explain their selective ability to cross present cellular antigens to both CD4+ and CD8+ T cells.

scholarly journals Comparison of In Vivo and Ex Vivo MRI for the Detection of Structural Abnormalities in a Mouse Model of Tauopathy

2017 ◽  
Vol 11 ◽  
Author(s):  
Holly E. Holmes ◽  
Nick M. Powell ◽  
Da Ma ◽  
Ozama Ismail ◽  
Ian F. Harrison ◽  
...  
Keyword(s):  
Mouse Model ◽  
Ex Vivo ◽  
Structural Abnormalities

scholarly journals Targeted In Situ Gene Correction of DysfunctionalAPOEAlleles to Produce Atheroprotective Plasma ApoE3 Protein

2012 ◽  
Vol 2012 ◽  
pp. 1-16 ◽  
Author(s):  
Ioannis Papaioannou ◽  
J. Paul Simons ◽  
James S. Owen
Keyword(s):  
Ex Vivo ◽  
Strand Break ◽  
Gene Correction ◽  
Cvd Risk ◽  
Transfer Strategies ◽  
Technical Advances ◽  
The Common ◽  
Induced Pluripotent

Cardiovascular disease is the leading worldwide cause of death. Apolipoprotein E (ApoE) is a 34-kDa circulating glycoprotein, secreted by the liver and macrophages with pleiotropic antiatherogenic functions and hence a candidate to treat hypercholesterolaemia and atherosclerosis. Here, we describe atheroprotective properties of ApoE, though also potential proatherogenic actions, and the prevalence of dysfunctional isoforms, outline conventional gene transfer strategies, and then focus on gene correction therapeutics that can repair defectiveAPOEalleles. In particular, we discuss the possibility and potential benefit of applying in combination two technical advances to repair aberrantAPOEgenes: (i) an engineered endonuclease to introduce a double-strand break (DSB) in exon 4, which contains the common, but dysfunctional,ε2 andε4 alleles; (ii) an efficient and selectable template for homologous recombination (HR) repair, namely, an adeno-associated viral (AAV) vector, which harbours wild-typeAPOEsequence. This technology is applicable ex vivo, for example to target haematopoietic or induced pluripotent stem cells, and also for in vivo hepatic gene targeting. It is to be hoped that such emerging technology will eventually translate to patient therapy to reduce CVD risk.

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