Name: a multimodal imaging approach based on micro-ct and fluorescence molecular tomography for longitudinal assessment of bleomycin-induced lung fibrosis in mice
Uploaded: 2018-04-13T12:30:00
Description: Watch this Scientific Journal Video about A Multimodal Imaging Approach Based on Micro-CT and Fluorescence Molecular Tomography for Longitudinal Assessment of Bleomycin-Induced Lung Fibrosis in Mice a

The authors would like to thank Dr. Daniela Pompilio and Roberta Ciccimarra for technical help.

All animal experiments described herein were approved by the intramural animal-welfare committee for animal experimentation of Chiesi Farmaceutici and ERASMUS MC under protocol number: EMC 3349 (138-14-07) complying with the European Directive 2010/63 UE, Italian D.Lgs 26/2014 and the revised &#34;Guide for the Care and Use of Laboratory Animals&#34;23.
NOTE: Prior to use, female inbred C57Bl/6 (7-8 weeks old) mice were acclimatized for at least 7 days to the local viva...

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Despite many research groups focusing on developing new drugs to treat IPF, at the moment only two are available for patients. There is an urgent medical need to find more effective therapies7 because only lung transplantationis able to prolong survival of 4-5 years26. The essential prerequisite for translational medicine and development of new drugs is the availability of an animal model that mimics the features of IPF and in which interventional studies are predictive of ...

Idiopathic pulmonary fibrosis (IPF) is chronic lung disease with progressive decrease of lung functions that is unfortunately often fatal within four years of diagnosis1. The major features of IPF are extracellular matrix deposition and fibroblast proliferation, but the pathogenesis is not yet fully understood. The most supported hypothesis is that multiple cycles of lung injuries cause the destruction of alveolar epithelial cells that leads to alteration of the mesenchymal cell cycle proliferation, exaggerated accumulation of fibroblasts and myofibroblasts, and increased matrix production. Mediators involved in these processes such as matrix metalloproteinases (MMPs) have been found dysregulated in fibrosis development either in human IPF or in bleomycin-induced animal models. The uncontrolled MMP production leads to an unbalanced collagen deposition within the lung interstitium and alveolar space, mimicking abnormal wound repair1,2.
One of the main obstacles for drug discovery and development is the availability of accessible mouse models that mimic human pathogenesis and the disease phenotype. Different agents have been used to induce lung fibrosis in animal models: irradiation damage, administration of asbestos and silica, administration of fibrinogenic cytokines and bleomycin3,4; however bleomycin is the most used in mice, rats, guinea pigs, hamsters, rabbits5 or in large animals (non-human primates, horses, dogs and ruminants)6,7. Bleomycin is an antibiotic made by the bacterium Streptomyces verticillus8 and is used as an anti-cancer agent9. Pulmonary fibrosis is a common side effect of the drug and for this reason, it is used in experimental animal models to induce pulmonary fibrosis.
In bleomycin-induced lung fibrosis models, the fibrotic lesions occur 14 - 21 days after bleomycin administration. In the presented work, we used a new protocol to induce lung fibrosis in mice by double bleomycin intratracheal instillation. The bleomycin mouse model is very time consuming because new drugs need to be evaluated on established fibrotic lesions, and tested to distinguish their anti-fibrotic effects from anti-inflammatory effects.
Biochemical determination of collagen content, morphometrical and histological analysis were based on post mortem analysis, limiting the possibility to follow the pathogenesis of the disease in the same animal. Although these parameters were considered a gold standard for fibrosis evaluation, they did not provide any temporal or spatial distribution of the fibrotic lesion and preclude a way to investigate the process of disease progression.10
Recently, non-invasive imaging technologies have been applied to monitor airway remodeling, inflammation, and fibrosis progression in murine models: Magnetic Resonance Imaging (MRI), Micro Computer Tomography (Micro-CT), Fluorescence Molecular Tomography (FMT) and Bioluminescent (BLI)11,12,13,14,15,16,17,18,19,20,21. We propose a non-invasive imaging approach to monitor longitudinally lung fibrosis progression by FMT and Micro-CT at different time-points after a bleomycin challenge22.
Many pathways are involved in the establishment and development of fibrosis, and not much is known. Only a deeper understanding of these processes could translate to more drug targets that may transfer into the clinic. The ability to longitudinally monitor MMP activation by fluorescence molecular tomography coupled to the detection of lung parenchymal changes by Micro-CT might be used in the future to access the clinical response to treatment.

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			<td height="24" style="height:24px;width:328px;">Penn-century model DP-4M Dry power insufflator</td>
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			<td>DPM-EXT</td>
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a multimodal imaging approach based on micro-ct and fluorescence molecular tomography for longitudinal assessment of bleomycin-induced lung fibrosis in mice

Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease characterized by the progressive and irreversible destruction of lung architecture, which causes significant deterioration in lung function and subsequent death from respiratory failure.
The pathogenesis of IPF in experimental animal models has been induced by bleomycin administration. In this study, we investigate an IPF-like mouse model induced by a double intratracheal bleomycin instillation. Standard histological assessments used for studying lung fibrosis are invasive terminal procedures. The goal of this work is to monitor lung fibrosis through noninvasive imaging techniques such as Fluorescent Molecular Tomography (FMT) and Micro-CT. These two technologies validated with histology findings could represent a revolutionary functional approach for real time non-invasive monitoring of IPF disease severity and progression. The fusion of different approaches represents a step further for understanding the IPF disease, where the molecular events occurring in a pathological condition can be observed with FMT and the subsequent anatomical changes can be monitored by Micro-CT.

Spontaneous resolution of the lung fibrosis lesions observed three weeks after single bleomycin administration and moderate structural changes highlight the limits of this model. Only preventive treatment could be performed due to the narrow therapeutic window that does not represent clinical practice17.
Here, we demonstrate that our protocol of double bleomycin intratracheal instillation is able to devel...

Watch this Scientific Journal Video about A Multimodal Imaging Approach Based on Micro-CT and Fluorescence Molecular Tomography for Longitudinal Assessment of Bleomycin-Induced Lung Fibrosis in Mice a

A Multimodal Imaging Approach Based on Micro-CT and Fluorescence Molecular Tomography for Longitudinal Assessment of Bleomycin-Induced Lung Fibrosis in Mice

We describe a non-invasive multimodal imaging approach based on Micro-CT and fluorescence molecular tomography for longitudinal assessment of the mouse lung fibrosis model induced by double intratracheal instillation of bleomycin.

Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease characterized by the progressive and irreversible destruction of lung architecture, which ...

The overall goal of this study on an IPF-like mouse model is to longitudinally monitor lung fibrosis with non-invasive imaging techniques such as FMT and micro-CT. This method can advance what sequence in regarding this specific molecular events occurring in lung fibrosis. The main advantage of this techniques is that anatomical changes of specific molecular events can be monitored noninvasively.The applications of this technologies extend towards therapy of IPF pathology because the fusion of FMT and micro-CT represents the possibility to study disease progression and new therapy evaluation. Start by positioning an anesthetized mouse on the intubation platform hanging it by its incisors placed on the wire. Turn on the laryngoscope, then take a pair of blunt-ended forceps and gently open the mouth.Place the laryngoscope blade towards the back of the mouth until the opening of the trachea is visualized. Use the other hand to insert the delivery tube connected to the end of the PE tubing into the trachea. Rotate the three-way valve to deliver 50 microliters of bleomycin.After installation quickly remove the tube from the trachea to prevent suffocation. Hold the mice upright for a few seconds. For probe injection take the mouse by the tail and allow it to grip the cage lid.Then grasp across the shoulders and hold the mouse by the scruff. Immerse the tail in a beaker of warm water to cause the veins to swell for easier visualization and needle insertion. Find the two caudal veins on either side of the tail, not the artery on the underside of the tail.Insert the 26 gauged needle of the MMP loaded 1 milliliter syringe into the vein and inject the MMP probe solution at 10 milliliters per kilogram of body weight. To begin initialize the date acquisition software and open a new study in the database for the experiment. Before starting the imaging, transfer the shaved and anesthetized mouse to the middle of imaging cassette.Keep the mouse flat, secure, and gently compressed against both windows of the imaging cassette. Then in the scanning window of the software, click on Subject"and then preview to see the live image. Draw the scan region sufficiently large so that the tissues surrounding the anticipated area of fluorescence is captured.Once the ROY is drawn, click Add to Reconstruction Queue"and then click Scan"to image the mouse. When the image acquisition is complete, remove the mouse from the imaging cassette and place back into the home cage. Quantify the picomoles of fluorescence using the ROY tool and reduce the ROY around 700 to 800 cubed millimeters on the signal coming from lung region.Repeat imaging every seven days or as required. To image by micro-CT, first turn on the micro-CT by pressing the green power button and launch the software to warm up the x-ray source. Create the database by clicking on New Database"and build the browser based on the number of mice in the experiment.Before starting the scan, select acquisition parameters in the software control window as follows. Set x-ray tube voltage to 90 kilovolts, the CT x-ray tube current to 160 micro angstroms, live x-ray tube current to 80 micro angstroms, and the field of view to 36 millimeters. Select no Gating Technique"and High Resolution"and four minutes for scan technique.After anesthetizing the mouse by inhalation of 3%isoflurane, place it onto the animal bed inserted into the small bore with a nose cone providing a constant supply of anesthetic. Immobilize the paws with tape on the bed to allow the chest to be exposed. Next side the instrument door closed and turn on live mode to see the mouse position in real time by pressing the capture button.Move the animal bed to align the chest with field of view using the buttons on the CT instrument. Once the mouse is centered, rotate the gantry to confirm optimal bed position by selecting 90 degrees and clicking Set"Then click the CT scan button. Click Yes"to the message that informs that the x-ray source will be turned on.After imaging place the animals back into the cage and monitor until fully recovered from anesthesia. Repeat imaging every seven days or as required. Staining with Masson's trichrome reveals the morphology in the lung tissue.Pictures of the bleomycin treated group showed a pronounced pattern of fibrosis starting from day seven mainly as single fibrotic masses and progressed at day 14 to confluent conglomerates of substituted collagen and remained unaltered until day 21. Repeated micro-CT imaging allowed for quantification and segmentation of airways in fibrosis lung lobes. The airways were divided into a central and distal part.The distal part of the airways is the intrapulmonary tract and splits into the lung lobes as the right cranial lobe, right middle lobe, right caudal lobe, right accessory lobe, and left lung. Quantification of airway radius showed increased radius in the bleomycin treated mice compared to saline controls. Each point represents the mean standard error of the mean of five animals for a total of 30 mice.Total lung fibrosis quantification of vehicle or bleomycin-treated mice at different time points demonstrates that these micro-CT parameters agree with histological findings. Once mastered this technique can be performed for drug screening routinely. After its development this technique paved the way for researcher in the field of lung fibrosis disease to explore efficacies of the repeating treatment in IPF-like animal models.

a-multimodal-imaging-approach-based-on-micro-ct-fluorescence

Research

JoVE Journal

Immunology and Infection

An Orthotopic Model of Serous Ovarian Cancer in Immunocompetent Mice for in vivo Tumor Imaging and Monitoring of Tumor Immune Responses

Background: Ovarian cancer is generally diagnosed at an advanced stage where the case/fatality ratio is high and thus remains the most lethal of all gynecologic malignancies among US women 1,2,3. Serous tumors are the most widespread forms of ovarian cancer and 4,5 the Tg-MISIIR-TAg transgenic represents the only mouse model that spontaneously develops this type of tumors. Tg-MISIIR-TAg mice express SV40 transforming region under control of the Mullerian Inhibitory Substance type II Receptor (MISIIR) gene promoter 6. Additional transgenic lines have been identified that express the SV40 TAg transgene, but do not develop ovarian tumors. Non-tumor prone mice exhibit typical lifespan for C57Bl/6 mice and are fertile. These mice can be used as syngeneic allograft recipients for tumor cells isolated from Tg-MISIIR-TAg-DR26 mice.
Objective: Although tumor imaging is possible 7, early detection of deep tumors is challenging in small living animals. To enable preclinical studies in an immunologically intact animal model for serous ovarian cancer, we describe a syngeneic mouse model for this type of ovarian cancer that permits in vivo imaging, studies of the tumor microenvironment and tumor immune responses.
Methods: We first derived a TAg+ mouse cancer cell line (MOV1) from a spontaneous ovarian tumor harvested in a 26 week-old DR26 Tg-MISIIR-TAg female. Then, we stably transduced MOV1 cells with TurboFP635 Lentivirus mammalian vector that encodes Katushka, a far-red mutant of the red fluorescent protein from sea anemone Entacmaea quadricolor with excitation/emission maxima at 588/635 nm 8,9,10. We orthotopically implanted MOV1Kat in the ovary 11,12,13,14 of non-tumor prone Tg-MISIIR-TAg female mice. Tumor progression was followed by in vivo optical imaging and tumor microenvironment was analyzed by immunohistochemistry.
Results: Orthotopically implanted MOV1Kat cells developed serous ovarian tumors. MOV1Kat tumors could be visualized by in vivo imaging up to three weeks after implantation (fig. 1) and were infiltrated with leukocytes, as observed in human ovarian cancers 15 (fig. 2).
Conclusions: We describe an orthotopic model of ovarian cancer suitable for in vivo imaging of early tumors due to the high pH-stability and photostability of Katushka in deep tissues. We propose the use of this novel syngeneic model of serous ovarian cancer for in vivo imaging studies and monitoring of tumor immune responses and immunotherapies.

An Orthotopic Model of Serous Ovarian Cancer in Immunocompetent Mice for in vivo Tumor Imaging and Monitoring of Tumor Immune Responses

To study in vivo tumor growth and tumor microenvironment, we used a syngeneic and orthotopic mouse model of ovarian cancer in immunocompetent animals. We transduced a mouse tumor cell line (MOV1) with Katushka fluorescent protein (MOV1KAT) and here we show its orthotopic implantation in ovary and in vivo imaging.

Background: Ovarian cancer is generally diagnosed at an advanced stage where the case/fatality ratio is high and thus remains the most lethal of all ...

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

There is growing concern about the relevance of in vitro antimicrobial susceptibility tests when applied to isolates of P. aeruginosa from cystic fibrosis (CF) patients. Existing methods rely on single or a few isolates grown aerobically and planktonically. Predetermined cut-offs are used to define whether the bacteria are sensitive or resistant to any given antibiotic1. However, during chronic lung infections in CF, P. aeruginosa populations exist in biofilms and there is evidence that the environment is largely microaerophilic2. The stark difference in conditions between bacteria in the lung and those during diagnostic testing has called into question the reliability and even relevance of these tests3.
 Artificial sputum medium (ASM) is a culture medium containing the components of CF patient sputum, including amino acids, mucin and free DNA. P. aeruginosa growth in ASM mimics growth during CF infections, with the formation of self-aggregating biofilm structures and population divergence4,5,6. The aim of this study was to develop a microtitre-plate assay to study antimicrobial susceptibility of P. aeruginosa based on growth in ASM, which is applicable to both microaerophilic and aerobic conditions.
 An ASM assay was developed in a microtitre plate format. P. aeruginosa biofilms were allowed to develop for 3 days prior to incubation with antimicrobial agents at different concentrations for 24 hours. After biofilm disruption, cell viability was measured by staining with resazurin. This assay was used to ascertain the sessile cell minimum inhibitory concentration (SMIC) of tobramycin for 15 different P. aeruginosa isolates under aerobic and microaerophilic conditions and SMIC values were compared to those obtained with standard broth growth. Whilst there was some evidence for increased MIC values for isolates grown in ASM when compared to their planktonic counterparts, the biggest differences were found with bacteria tested in microaerophilic conditions, which showed a much increased resistance up to a &gt;128 fold, towards tobramycin in the ASM system when compared to assays carried out in aerobic conditions.
 The lack of association between current susceptibility testing methods and clinical outcome has questioned the validity of current methods3. Several in vitro models have been used previously to study P. aeruginosa biofilms7, 8. However, these methods rely on surface attached biofilms, whereas the ASM biofilms resemble those observed in the CF lung9 . In addition, reduced oxygen concentration in the mucus has been shown to alter the behavior of P. aeruginosa2 and affect antibiotic susceptibility10. Therefore using ASM under microaerophilic conditions may provide a more realistic environment in which to study antimicrobial susceptibility.

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

Current diagnostic antimicrobial susceptibility testing relies on the planktonic growth of isolates in nutrient rich, aerobic conditions. Here, we employ an alternative artificial sputum medium to study antimicrobial susceptibility of Pseudomonas aeruginosa biofilms under both aerobic and microaerophilic conditions more representative of the cystic fibrosis lung.

There  is growing concern about the relevance of in vitro antimicrobial  susceptibility tests when applied to isolates of P. aeruginosa from  cystic ...

4D Multimodality Imaging of Citrobacter rodentium Infections in Mice

This protocol outlines the steps required to longitudinally monitor a bioluminescent bacterial infection using composite 3D diffuse light imaging tomography with integrated &mu;CT (DLIT-&mu;CT) and the subsequent use of this data to generate a four dimensional (4D) movie of the infection cycle. To develop the 4D infection movies and to validate the DLIT-&mu;CT imaging for bacterial infection studies using an IVIS Spectrum CT, we used infection with bioluminescent C. rodentium, which causes self-limiting colitis in mice. In this protocol, we outline the infection of mice with bioluminescent C. rodentium and non-invasive monitoring of colonization by daily DLIT-&mu;CT imaging and bacterial enumeration from feces for 8 days.
The use of the IVIS Spectrum CT facilitates seamless co-registration of optical and &mu;CT scans using a single imaging platform. The low dose &mu;CT modality enables the imaging of mice at multiple time points during infection, providing detailed anatomical localization of bioluminescent bacterial foci in 3D without causing artifacts from the cumulative radiation. Importantly, the 4D movies of infected mice provide a powerful analytical tool to monitor bacterial colonization dynamics in vivo.

4D Multimodality Imaging of Citrobacter rodentium Infections in Mice

Multi-modality imaging is a valuable approach for studying bacterial colonization in small animal models. This protocol outlines infection of mice with bioluminescent Citrobacter rodentium and the longitudinal monitoring of bacterial colonization using composite 3D diffuse light imaging tomography with &mu;CT imaging to create a 4D movie of C. rodentium infection.

This protocol outlines the steps required to  longitudinally monitor a bioluminescent bacterial infection using composite 3D diffuse light imaging ...

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model itself. Early phases of inflammation and infection have been widely studied by using the Pseudomonas aeruginosa agar-beads mouse model, while only few reports have focused on the long-term chronic infection in vivo. The main challenge for long term chronic infection remains the low bacterial burden by P. aeruginosa and the low percentage of infected mice weeks after challenge, indicating that bacterial cells are progressively cleared by the host.
This paper presents a method for obtaining efficient long-term chronic infection in mice. This method is based on the embedding of the P. aeruginosa clinical strains in the agar-beads in vitro, followed by intratracheal instillation in C57Bl/6NCrl mice. Bilateral lung infection is associated with several measurable read-outs including weight loss, mortality, chronic infection, and inflammatory response. The P. aeruginosa RP73 clinical strain was preferred over the PAO1 reference laboratory strain since it resulted in a comparatively lower mortality, more severe lesions, and higher chronic infection. P. aeruginosa colonization may persist in the lung for over three months. Murine lung pathology resembles that of CF patients with advanced chronic pulmonary disease.
This murine model most closely mimics the course of the human disease and can be used both for studies on the pathogenesis and for the evaluation of novel therapies.

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

We describe the agar-beads method to establish persistent long-term chronic Pseudomonas aeruginosa airway infection in the mouse model.

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model ...

Imaging Neutrophils and Monocytes in Mesenteric Veins by Intravital Microscopy on Anaesthetized Mice in Real Time

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in vivo is crucial for understanding the molecular basis of leukocyte transendothelial migration and interaction with vascular endothelium. One powerful approach involves intravital microscopy on transgenic mice expressing fluorescent proteins in cells of interest.
Here we present a protocol for imaging monocytes and neutrophils in the CX3CR1gfp/wt mouse i.v. injected with orange dye-labeled neutrophils with an inverted confocal microscope. Time-lapse movies gathered from 30 min&#160;to several hours of imaging allow the analysis of leukocyte behavior in mesenteric veins under both steady state and inflammatory conditions. We also describe the steps to locally induce blood vessel inflammation with TLR2/TLR1 agonist Pam3SK4 and monitor the subsequent recruitment of neutrophils and monocytes.
The presented technique can also be used to monitor other populations of leukocytes and investigate molecules implicated in leukocyte recruitment or trafficking using other stimuli or transgenic mice.

We detail a protocol to monitor the behavior of neutrophils and monocytes in mesenteric veins under steady state and inflammatory conditions using intravital confocal microscopy on anaesthetized mice.

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in ...

A Simple and Efficient Approach to Construct Mutant Vaccinia Virus Vectors

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been successfully employed for editing genomes of various organisms. Here we describe a protocol for editing the vaccinia virus (VV) genome in the cytoplasm of VV-infected CV-1 cells using the RNA-guided Cas9. RNA-guided Cas9 induces double-stranded DNA breaks facilitating homologous recombination efficiently and specifically in the targeted site of VV and a transgene can be incorporated into these sites by homologous recombination. By using a site-specific homologous vector with transgene(s), a N1L gene-deleted VV with the red fluorescence protein (RFP) gene incorporated in this region was generated with a successful recombination efficiency 10 times greater than that obtained from the conventional homologous recombination method. This protocol demonstrates successful use of RNA-guided Cas9 system to generate mutant VVs with enhanced efficiency.

Vaccinia virus (VV) has been widely used in biomedical research and the improvement of human health. This article describes a simple, highly efficient method to edit the VV genome using a CRISPR-Cas9 system.

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been ...

A Fluorescence-based Lymphocyte Assay Suitable for High-throughput Screening of Small Molecules

High-throughput screening (HTS) is currently the mainstay for the identification of chemical entities capable of modulating biochemical reactions or cellular processes. With the advancement of biotechnologies and the high translational potential of small molecules, a number of innovative approaches in drug discovery have evolved, which explains the resurgent interest in the use of HTS. The oncology field is currently the most active research area for drug screening, with no major breakthrough made for the identification of new immunomodulatory compounds targeting transplantation-related complications or autoimmune ailments. Here, we present a novel in vitro murine fluorescent-based lymphocyte assay easily adapted for the identification of new immunomodulatory compounds. This assay uses T or B cells derived from a transgenic mouse, in which the Nur77 promoter drives GFP expression upon T- or B-cell receptor stimulation. As the GFP intensity reflects the activation/transcriptional activity of the target cell, our assay defines a novel tool to study the effect of given compound(s) on cellular/biological responses. For instance, a primary screening was performed using 4,398 compounds in the absence of a &#34;target hypothesis&#34;, which led to the identification of 160 potential hits displaying immunomodulatory activities. Thus, the use of this assay is suitable for drug discovery programs exploring large chemical libraries prior to further in vitro/in vivo validation studies.

We present in the current study a novel fluorescence-based assay using lymphocytes derived from a transgenic mouse. This assay is suitable for high-throughput screening (HTS) of small molecules endowed with the capacity of either inhibiting or promoting lymphocyte activation.

High-throughput screening (HTS) is currently the mainstay for the identification of chemical entities capable of modulating biochemical reactions or ...

Fluorescence-based Neuraminidase Inhibition Assay to Assess the Susceptibility of Influenza Viruses to The Neuraminidase Inhibitor Class of Antivirals

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against currently circulating strains. In addition to their use in treating seasonal influenza, the NA inhibitors have been stockpiled by a number of countries for use in the event of a pandemic. It is therefore important to monitor the susceptibility of circulating influenza viruses to this class of antivirals. There are different types of assays that can be used to assess the susceptibility of influenza viruses to the NA inhibitors, but the enzyme inhibition assays using either a fluorescent substrate or a chemiluminescent substrate are the most widely used and recommended. This protocol describes the use of a fluorescence-based assay to assess influenza virus susceptibility to NA inhibitors. The assay is based on the NA enzyme cleaving the 2&#8242;-(4-Methylumbelliferyl)-&#945;-D-N-acetylneuraminic acid (MUNANA) substrate to release the fluorescent product 4-methylumbelliferone (4-MU). Therefore, the inhibitory effect of an NA inhibitor on the influenza virus NA is determined based on the concentration of the NA inhibitor that is required to reduce 50% of the NA activity, given as an IC50 value.

We describe the use of a phenotypic fluorescence-based neuraminidase inhibition assay to assess the susceptibility of influenza A and B viruses to the neuraminidase inhibitor class of antivirals.

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against ...

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by fluorescence or bioluminescence. We utilize BlaC, an enzyme expressed constitutively by all M. tuberculosis strains. REF allows rapid quantification of bacteria in lungs of infected mice. The same group of mice can be imaged at many time points, greatly reducing costs, enumerating bacteria more quickly, allowing novel observations in host-pathogen interactions, and increasing statistical power, since more animals per group are readily maintained. REF is extremely sensitive due to the catalytic nature of the BlaC enzymatic reporter and specific due to the custom flourescence resonance energy transfer (FRET) or fluorogenic substrates used. REF does not require recombinant strains, ensuring normal host-pathogen interactions. We describe the imaging of M. tuberculosis infection using a FRET substrate with maximal emission at 800 nm. The wavelength of the substrate allows sensitive deep tissue imaging in mammals. We will outline aerosol infection of mice with M. tuberculosis, anesthesia of mice, administration of the REF substrate, and optical imaging. This method has been successfully applied to evaluating host-pathogen interactions and efficacy of antibiotics targeting M. tuberculosis.

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

We describe the optical imaging of mice infected with Mycobacterium tuberculosis (M. tuberculosis) using reporter enzyme fluorescence (REF). This protocol facilitates the sensitive and specific detection of M. tuberculosis in pre-clinical animal models for pathogenesis, therapeutics and vaccine research.

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by ...

Assessment of the Cytotoxic and Immunomodulatory Effects of Substances in Human Precision-cut Lung Slices

Respiratory diseases in their broad diversity need appropriate model systems to understand the underlying mechanisms and enable development of new therapeutics. Additionally, registration of new substances requires appropriate risk assessment with adequate testing systems to avoid the risk of individuals being harmed, for example, in the working environment. Such risk assessments are usually conducted in animal studies. In view of the 3Rs principle and public skepticism against animal experiments, human alternative methods, such as precision-cut lung slices (PCLS), have been evolving. The present paper describes the ex vivo technique of human PCLS to study the immunomodulatory potential of low-molecular-weight substances, such as ammonium hexachloroplatinate (HClPt). Measured endpoints include viability and local respiratory inflammation, marked by altered secretion of cytokines and chemokines. Pro-inflammatory cytokines, tumor necrosis factor alpha (TNF-&#945;), and interleukin 1 alpha (IL-1&#945;) were significantly increased in human PCLS after exposure to a sub-toxic concentration of HClPt. Even though the technique of PCLS has been substantially optimized over the past decades, its applicability for the testing of immunomodulation is still in development. Therefore, the results presented here are preliminary, even though they show the potential of human PCLS as a valuable tool in respiratory research.

In view of the 3Rs principle, respiratory models as alternatives to animal studies are evolving. Especially for risk assessment of respiratory substances, there is a lack of appropriate assays. Here, we describe the use of human precision-cut lung slices for the assessment of airborne substances.