Name: intraperitoneal glucose tolerance test, measurement of lung function, and fixation of the lung to study the impact of obesity and impaired metabolism on pulmonary outcomes
Uploaded: 2018-03-15T13:00:00
Description: Watch this Scientific Journal Video about Intraperitoneal Glucose Tolerance Test, Measurement of Lung Function, and Fixation of the Lung to Study the Impact of Obesity and Impaired Metabolism on Pulmo

The experiments were supported by the Marga and Walter Boll-Stiftung, Kerpen, Germany; Project 210-02-16 (MAAA), Project 210-03-15 (MAAA) and by the German Research Foundation (DFG; AL1632-02; MAAA), Bonn, Germany; Center of Molecular Medicine Cologne (CMMC; University Hospital Cologne; Career Advancement Program; MAAA), K&#246;ln Fortune (Faculty of Medicine, University of Cologne; KD).

All animal procedures were conducted in compliance with protocols approved by local government authorities (Land NRW, AZ: 2012.A424), and were in accordance with the German animal welfare law and the regulations on the welfare of animals used for experiments or for other scientific purposes. Since lung function analysis may affect lung structure and therefore subsequent histological analyzes, the measurement of Res and Cdyn and the preparation and fixation of the lung for histomorphometry should be performed in different...

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This report provides three protocols for three different methods to analyze the impact of obesity on glucose metabolism and pulmonary outcomes. First, the glucose tolerance test offers the opportunity to analyze intracellular glucose uptake and can be indicative of insulin resistance. Second, whole body plethysmography is a technique to measure lung function and is thereby helpful to test the efficacy of novel treatments. Third, a standardized fixation protocol is essential for quantitative morphometric analysis to asses...

According to the World Health Organization (WHO) in 2008, more than 1.4 billion adults, aged 20 and older, were overweight with a body mass index (BMI) greater than or equal to 25; further, over 200 million men and nearly 300 million women were obese (BMI&#8805;30)1. Obesity and metabolic syndrome are major risk factors for a multitude of diseases. While obesity and concomitant increased white adipose tissue mass has been intimately linked to type 2 diabetes2,3, cardio-vascular diseases including coronary heart disease (CHD), heart failure (HF), atrial fibrillation4 and osteoarthritis5, their functional roles in the pathogenesis of respiratory disorders remain poorly understood. However, epidemiological studies have demonstrated that obesity is strongly associated with chronic respiratory conditions, including exertional dyspnea, obstructive sleep apnea syndrome (OSAS), obesity hypoventilation syndrome (OHS), chronic obstructive pulmonary disease (COPD), pulmonary embolism, aspiration pneumonia and bronchial asthma6,7,8,9. Potential mechanisms linking obesity and perturbed metabolism, e.g., insulin resistance and type II diabetes, to the pathogenesis of chronic lung disease not only comprise mechanical and physical consequences of weight gain on ventilation but also induce a chronic subacute inflammatory state10,11. The rise of obesity and lung diseases during the last decade, coupled with the lack of effective preventive strategies and therapeutic approaches, highlights the need to investigate the molecular mechanisms to define new avenues to manage obesity-related lung diseases.
Here, we describe three standard tests, which are important basics to investigate obesity and its impact on lung structure and function in mouse models: (1) intraperitoneal glucose tolerance (ipGTT) (2) measurement of airway resistance (Res) and respiratory system compliance (Cdyn); and (3) preparation and fixation of the lung for subsequent quantitative histological assessment. The ipGTT is a robust screening test to measure glucose uptake, and thus the effect of obesity on metabolism. The simplicity of the method allows good standardization, and therefore the comparability of results between laboratories. More sophisticated methods, such as hyperglycemic clamps or studies on isolated islets, can be used for detailed analysis of the metabolic phenotype12. Here we assess glucose tolerance to define an obesity-associated state of systemic and metabolic disorder as the basis for further studies on a pulmonary outcome. To assess the effect of obesity and metabolic disorder on lung function, we measured airway resistance (Res) and respiratory system compliance (Cdyn). To characterize lung disease, unrestrained as well as restrained methods for assessment of lung function are available. Unrestrained plethysmography in freely moving animals mimics a natural state, reflecting breathing patterns; in contrast, invasive methods, such as input impedance measurement of Res and cDyn in deeply anesthetized mice to assess dynamic lung mechanics, are more accurate13. Since chronic respiratory conditions are reflected by histologic alterations of the lung tissue, proper lung fixation for further analysis is imminent. The choice of the method of tissue fixation and preparation depends on the compartment of the lung which will be studied, for example, conducting airways or lung parenchyma14. Here, we describe a method that allows qualitative and quantitative assessment of the conducting airways to study the effect of obesity on asthma development.

<table>
	<tbody>
		<tr>
			<td>GlucoMen LX</td>
			<td>A.Menarini diagnostics, Firneze, Italy</td>
			<td>38969</td>
			<td>blood glucose meter</td>
		</tr>
		<tr>
			<td>GlucoMen LX Sensor</td>
			<td>A.Menarini diagnostics, Firneze, Italy</td>
			<td>39765</td>
			<td>Test stripes</td>
		</tr>
		<tr>
			<td>Glucose 20%</td>
			<td>B. Braun, Melsung, Germany</td>
			<td>2356746</td>
			<td></td>
		</tr>
		<tr>
			<td>FinePointe Software</td>
			<td>DSI, MC s&#180;Hertogenbosch, Netherlands</td>
			<td>601-1831-002</td>
			<td></td>
		</tr>
		<tr>
			<td>FinePointe RC Single Site Mouse Table</td>
			<td>DSI, MC s&#180;Hertogenbosch, Netherlands</td>
			<td>601-1831-001</td>
			<td></td>
		</tr>
		<tr>
			<td>FPRC Controller</td>
			<td>DSI, MC s&#180;Hertogenbosch, Netherlands</td>
			<td>601-1075-001</td>
			<td></td>
		</tr>
		<tr>
			<td>FPRC Aerosol Block</td>
			<td>DSI, MC s&#180;Hertogenbosch, Netherlands</td>
			<td>601-1106-001</td>
			<td></td>
		</tr>
		<tr>
			<td>Aerogen neb head-5.2-4um</td>
			<td>DSI, MC s&#180;Hertogenbosch, Netherlands</td>
			<td>601-2306-001</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>FST, British Columbia, Canada</td>
			<td>11065-07</td>
			<td></td>
		</tr>
		<tr>
			<td>Blunt scissors</td>
			<td>FST, British Columbia, Canada</td>
			<td>14105-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro scissors</td>
			<td>FST, British Columbia, Canada</td>
			<td>15000-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Perma-Hand 4-0</td>
			<td>Ethicon, Puerto Rico, USA</td>
			<td>736H</td>
			<td>Surgical suture</td>
		</tr>
		<tr>
			<td>Roti-Histofix 4%</td>
			<td>Roth</td>
			<td>P087.1</td>
			<td>4% Paraformaldehyd</td>
		</tr>
		<tr>
			<td>Ketaset</td>
			<td>Zoetis, Berlin, Germany</td>
			<td>10013389</td>
			<td>Ketamine</td>
		</tr>
		<tr>
			<td>Rompun 2%</td>
			<td>Bayer, Leverkusen, Germany</td>
			<td>770081</td>
			<td>Xylazine</td>
		</tr>
	</tbody>
</table>

intraperitoneal glucose tolerance test, measurement of lung function, and fixation of the lung to study the impact of obesity and impaired metabolism on pulmonary outcomes

Obesity and respiratory disorders are major health problems. Obesity is becoming an emerging epidemic with an expected number of over 1 billion obese individuals worldwide by 2030, thus representing a growing socioeconomic burden. Simultaneously, obesity-related comorbidities, including diabetes as well as heart and chronic lung diseases, are continuously on the rise. Although obesity has been associated with increased risk for asthma exacerbations, worsening of respiratory symptoms, and poor control, the functional role of obesity and perturbed metabolism in the pathogenesis of chronic lung disease is often underestimated, and underlying molecular mechanisms remain elusive. This article aims to present methods to assess the effect of obesity on metabolism, as well as lung structure and function. Here, we describe three techniques for mice studies: (1) assessment of intraperitoneal glucose tolerance (ipGTT) to analyze the effect of obesity on glucose metabolism; (2) measurement of airway resistance (Res) and respiratory system compliance (Cdyn) to analyze the effect of obesity on lung function; and (3) preparation and fixation of the lung for subsequent quantitative histological assessment. Obesity-related lung diseases are probably multifactorial, stemming from systemic inflammatory and metabolic dysregulation that potentially adversely influence lung function and the response to therapy. Therefore, a standardized methodology to study molecular mechanisms and the effect of novel treatments is essential.

Representative results of intraperitoneal glucose tolerance test (ipGTT) (Figure 4), lung function test (Figure 5), and representative images illustrating hematoxylin and eosin stained lungs (Figure 6).
The ipGTT was performed in obese mice (blue) after 7 weeks of high-fat-diet (HFD). Standard diet-fed mice served as controls (black). Obese...

Watch this Scientific Journal Video about Intraperitoneal Glucose Tolerance Test, Measurement of Lung Function, and Fixation of the Lung to Study the Impact of Obesity and Impaired Metabolism on Pulmo

Intraperitoneal Glucose Tolerance Test, Measurement of Lung Function, and Fixation of the Lung to Study the Impact of Obesity and Impaired Metabolism on Pulmonary Outcomes

The incidence of obesity is rising and increases the risk of chronic lung diseases. To establish the underlying mechanisms and preventive strategies, well-defined animal models are needed. Here, we provide three methods (glucose-tolerance-test, body plethysmography, and lung fixation) to study the effect of obesity on pulmonary outcomes in mice.

Obesity and respiratory disorders are major health problems. Obesity is becoming an emerging epidemic with an expected number of over 1 billion obese ...

The overall goal of the following three methods is to study the impact of obesity and metabolic disorders on lung structure and function. These methods can help to answer key questions in the respiratory research field about the impact of metabolic disorders on lung structure and lung function. The advantage of these techniques is that they can be combined to assess the functional role of metabolism on the pathogenesis of chronic lung diseases and to test different therapeutical approaches.The implications of these techniques extend beyond phenotyping to what's elucidating the specific functional role of metabolic targets in the pathogenesis of lung diseases. These methods can also be applied to the study of structure and function in both healthy animals with genetic modifications but also in animal models of COPD and asthma. Generally, individuals new to these methods will struggle as insertion of the tracheal tube, the insufflation of the lung and the administration of the deep anesthesia, they all require experience and training.We first had the idea for these methods when we discovered that early onset obesity in mice but also in humans were intimately linked to impact metabolism and to chronic lung diseases beyond infancy. To measure intraperitoneal glucose tolerance, gently restrain a 12-hour fasted adult mouse by the tail and use sterile scissors to incise the tail tip. Immediately apply a free-flowing drop of blood to the test strip of the blood glucose meter to measure the fasted blood glucose level.The test result will appear on the glucometer screen after four seconds. After measuring the glucose for each animal, label them individually with colored markers and weigh the animals. Then use a 27-gauge needle attached to a one-milliliter syringe to administer 100 microliters of glucose per ton grams of body weight to each animal via intraperitoneal injection and measure the blood glucose levels 15, 30, 60 and 120 minutes later with new test strips as just demonstrated.To assess the lung function, place an adult mouse in the supine position on a pad and apply ointment to the animal's eyes. After confirming a lack of response to toe pinch, wet the fur in the thyroid region with 70%ethanol and use forceps to lift the skin at the midline between the jugular notch at the sternum and the tuber symphysis of the mentum. Using blunt scissors, carefully make a one-centimeter skin incision in the tented skin to visualize the underlying subcutaneous adipose tissue and thyroid gland and expose the trachea with careful blunt separation of both thyroid lobes at the isthmus and dissection of the sternothyroid and sternothyroid muscles.Using sharp forceps, pass a 4-0 braided surgical suture between the trachea and the esophagus and carefully incise the trachea close the the larynx between the tracheal cartilages with microscissors. Intubate with a tracheal tube securing the tube with a surgical suture and move the animal to the heated bed of a body chamber. Connect the tracheal tube to the face plate and turn on the ventilator.Confirm movement of the thorax contemporaneously with the ventilation rate to confirm proper placement of the tracheal tube and watch the pressure signal on the computer screen. To control changes in the transpulmonary pressure during ventilation, insert an esophageal tube to the level of the lungs watching the screen to facilitate placement of the tube to where maximal pressure deflection and minimal heart artifacts can be observed. Begin the data acquisition according to the manufacturer's instructions and apply 10 microliters of PBS on the nebulizer.Start nebulization after five minutes of acclamation followed by a response phase of three minutes during which the airway resistance and respiratory system compliance are measured. Allow the animal to recover for three minutes before starting the next nebulization and follow the software instructions for stepwise application of increasing 10-microliter concentrations of methacholine on the ventilator. For quantitative histomorphometric analysis of the lung tissue, use blunt scissors to make a small incision in the diaphragm of an adult mouse and use curved blunt scissors to make a peristernal incision along the entire length of the rib cage to gently open the thorax.Lift the rib cage to expose the pleural cavity and remove the thymus. After removing the heart, use sharp forceps to prepare and expose the trachea in order to pass a 4-0 braided surgical suture between the trachea and the esophagus followed by careful incision of the trachea close to the larynx between the tracheal cartilages. Intubate with a 26-gauge intravenous cannula and use a fixative agent to inflate the lungs by pressure fixation at a constant pressure of 20 centimeters of water.After 30 minutes at room temperature, ligate the trachea and remove the cannula at the same time. Then carefully excise the lungs without damaging the tissue and store the lungs in fresh fixative agent at four degrees Celsius overnight. Obese mice demonstrate an increase in serum glucose levels 15 and 30 minutes after intraperitoneal glucose injection indicating an impaired cellular glucose uptake compared to standard diet fed mice.Invasive lung function analysis shows an up to 1.5-fold increase of airway resistance in obese, high-fat diet fed mice after stimulation with methacholine compared to control animals. Hematoxylin and eosin staining of lung parenchyma sections after intratracheal installation reveals multiple uninflated areas, thick alveolar septa and polygonal-shaped alveoli in lungs instilled with too little pressure. In contrast, too much instillation pressure leads to overinflated areas with destructed alveolar septa.Application of the appropriate amount of pressure during lung fixation however leads to a completely inflated lung with round-shaped alveoli. Once mastered, the intraperitoneal glucose tolerance tests can be completed in two and a half hour. The lung function tests can be completed in one hour, and the lung insufflation can be completed in under an hour.While attempting this procedure, it's important to remember that adapting the mice, training the investigator and working in a quiet and stress-reduced environment all are recommended for optimal data acquisition. After its development, this technique paved the way for researchers in the fields of metabolism and respiratory diseases to explore the mechanisms involved in and potention of treatment strategies of chronic lung disease using small animal models. After watching this video, you should have a good understanding how to investigate the impact of obesity and metabolic disorders on lung structure and function.Don't forget that working with animals or surgical instruments can cause injuries or skin reactions and that precautions such as wearing the appropriate personal protection equipment should always be taken while performing these procedures.

intraperitoneal-glucose-tolerance-test-measurement-lung-function

Research

JoVE Journal

Immunology and Infection

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 ...

Measurement of Antibody Effects on Cellular Function of Isolated Cardiomyocytes

Dilated cardiomyopathy (DCM) is one of the main causes for heart failure in younger adults1. Although genetic disposition and exposition to toxic substances are known causes for this disease in about one third of the patients, the origin of DCM remains largely unclear. In a substantial number of these patients, autoantibodies against cardiac epitopes have been detected and are suspected to play a pivotal role in the onset and progression of the disease2,3. The importance of cardiac autoantibodies is underlined by a hemodynamic improvement observed in DCM patients after elimination of autoantibodies by immunoadsorption3-5. A variety of specific antigens have already been identified2,3 and antibodies against these targets may be detected by immunoassays. However, these assays cannot discriminate between stimulating (and therefore functionally effective) and blocking autoantibodies. There is increasing evidence that this distinction is crucial6,7. It can also be assumed that the targets for a number of cardiotropic antibodies are still unidentified and therefore cannot be detected by immunoassays. Therefore, we established a method for the detection of functionally active cardiotropic antibodies, independent of their respective antigen. The background for the method is the high homology usually observed for functional regions of cardiac proteins in between mammals8,9. This suggests that cardiac antibodies directed against human antigens will cross-react with non-human target cells, which allows testing of IgG from DCM patients on adult rat cardiomyocytes. Our method consists of 3 steps: first, IgG is isolated from patient plasma using sepharose coupled anti-IgG antibodies obtained from immunoadsorption columns (PlasmaSelect, Teterow, Germany). Second, adult cardiomyocytes are isolated by collagenase perfusion in a Langendorff perfusion apparatus using a protocol modified from previous works10,11. The obtained cardiomyocytes are attached to laminin-coated chambered coverglasses and stained with Fura-2, a calcium-selective fluorescent dye which can be easily brought into the cell to observe intracellular calcium (Ca2+) contents12. In the last step, the effect of patient IgG on the cell shortening and Ca2+ transients of field stimulated cardiomyocytes is monitored online using a commercial myocyte calcium and contractility monitoring system (IonOptix, Milton, MA, USA) connected to a standard inverse fluorescent microscope.

The presented method offers a way to detect functional effective cardiotropic autoantibodies in the plasma of patients with dilated cardiomyopathy, irrespective of the specific antigen, by analysing the impact of isolated patient immunoglobulin on cellular shortening and intracellular calcium transients in isolated rat cardiomyocytes.

Dilated cardiomyopathy  (DCM) is one of the main causes for heart failure in younger adults1.  Although genetic disposition and exposition to toxic ...

In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

T cell exhaustion is a major factor in failed pathogen clearance during chronic viral infections. Immunoregulatory pathways, such as PD-1 and IL-10, are upregulated upon this ongoing antigen exposure and contribute to loss of proliferation, reduced cytolytic function, and impaired cytokine production by CD4 and CD8 T cells. In the murine model of LCMV infection, administration of blocking antibodies against these two pathways augmented T cell responses. However, there is currently no in vitro assay to measure the impact of such blockade on cytokine secretion in cells from human samples. Our protocol and experimental approach enable us to accurately and efficiently quantify the restoration of cytokine production by HIV-specific CD4 T cells from HIV infected subjects.
Here, we depict an in vitro experimental design that enables measurements of cytokine secretion by HIV-specific CD4 T cells and their impact on other cell subsets. CD8 T cells were depleted from whole blood and remaining PBMCs were isolated via Ficoll separation method. CD8-depleted PBMCs were then incubated with blocking antibodies against PD-L1 and/or IL-10R&#945; and, after stimulation with an HIV-1 Gag peptide pool, cells were incubated at 37 &#176;C, 5% CO2. After 48 hr, supernatant was collected for cytokine analysis by beads arrays and cell pellets were collected for either phenotypic analysis using flow cytometry or transcriptional analysis using qRT-PCR. For more detailed analysis, different cell populations were obtained by selective subset depletion from PBMCs or by sorting using flow cytometry before being assessed in the same assays. These methods provide a highly sensitive and specific approach to determine the modulation of cytokine production by antigen-specific T-helper cells and to determine functional interactions between different populations of immune cells.

In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

We developed an in vitro assay to investigate the role of immunoregulatory pathways in the regulation of cytokine secretion by HIV-specific CD4 T cells.

T cell exhaustion is a major factor in failed pathogen clearance during chronic viral infections. Immunoregulatory pathways, such as PD-1 and IL-10, are ...

Pseudomonas aeruginosa Induced Lung Injury Model

In order to study human acute lung injury and pneumonia, it is important to develop animal models to mimic various pathological features of this disease. Here we have developed a mouse lung injury model by intra-tracheal injection of bacteria Pseudomonas aeruginosa (P. aeruginosa or PA). Using this model, we were able to show lung inflammation at the early phase of injury. In addition, alveolar epithelial barrier leakiness was observed by analyzing bronchoalveolar lavage (BAL); and alveolar cell death was observed by Tunel assay using tissue prepared from injured lungs. At a later phase following injury, we observed cell proliferation required for the repair process. The injury was resolved 7 days from the initiation of P. aeruginosa injection. This model mimics the sequential course of lung inflammation, injury and repair during pneumonia. This clinically relevant animal model is suitable for studying pathology, mechanism of repair, following acute lung injury, and also can be used to test potential therapeutic agents for this disease.

Pseudomonas aeruginosa Induced Lung Injury Model

We have developed a mouse lung injury model by intra-tracheal injection of bacteria Pseudomonas aeruginosa. This model mimics lung injury during pneumonia and is clinically relevant.

In order to study human acute lung injury and pneumonia, it is important to develop animal models to mimic various pathological features of this disease. ...

Influenza Virus Propagation in Embryonated Chicken Eggs

Influenza infection is associated with about 36,000 deaths and more than 200,000 hospitalizations every year in the United States. The continuous emergence of new influenza virus strains due to mutation and re-assortment complicates the control of the virus and necessitates the permanent development of novel drugs and vaccines. The laboratory-based study of influenza requires a reliable and cost-effective method for the propagation of the virus. Here, a comprehensive protocol is provided for influenza A virus propagation in fertile chicken eggs, which consistently yields high titer viral stocks. In brief, serum pathogen-free (SPF) fertilized chicken eggs are incubated at 37 &#176;C and 55-60% humidity for 10 &#8211; 11 days. Over this period, embryo development can be easily monitored using an egg candler. Virus inoculation is carried out by injection of virus stock into the allantoic cavity using a needle. After 2 days of incubation at 37 &#176;C, the eggs are chilled for at least 4 hr at 4 &#176;C. The eggshell above the air sac and the chorioallantoic membrane are then carefully opened, and the allantoic fluid containing the virus is harvested. The fluid is cleared from debris by centrifugation, aliquoted and transferred to -80 &#176;C for long-term storage. The large amount (5-10 ml of virus-containing fluid per egg) and high virus titer which is usually achieved with this protocol has made the usage of eggs for virus preparation our favorable method, in particular for in vitro studies which require large quantities of virus in which high dosages of the same virus stock are needed.

Fertile chicken eggs are widely used to produce large amounts of human influenza A virus as they provide a convenient and cost-effective system to prove high yields of virus.

Influenza infection is associated with about 36,000 deaths and more than 200,000 hospitalizations every year in the United States. The continuous ...

An Optimized Protocol to Analyze Glycolysis and Mitochondrial Respiration in Lymphocytes

Lymphocytes respond to a variety of stimuli by activating intracellular signaling pathways, which in turn leads to rapid cellular proliferation, migration and differentiation, and cytokine production. All of these events are tightly linked to the energy status of the cell, and therefore studying the energy-producing pathways may give clues about the overall functionality of these cells. The extracellular flux analyzer is a commonly used device for evaluating the performance of glycolysis and mitochondrial respiration in many cell types. This system has been used to study immune cells in a few published reports, yet a comprehensive protocol optimized particularly for lymphocytes is lacking. Lymphocytes are fragile cells that survive poorly in ex vivo conditions. Oftentimes lymphocyte subsets are rare, and working with low cell numbers is inevitable. Thus, an experimental strategy that addresses these difficulties is required. Here, we provide a protocol that allows for rapid isolation of viable lymphocytes from lymphoid tissues, and for the analysis of their metabolic states in the extracellular flux analyzer. Furthermore, we provide results of experiments in which the metabolic activities of several lymphocyte subtypes at different cell densities were compared. These observations suggest that our protocol can be used to achieve consistent, well-standardized results even at low cell concentrations, and thus it may have broad applications in future studies focusing on the characterization of metabolic events in immune cells.

The study of metabolism is becoming increasingly relevant to immunological research. Here, we present an optimized method for measuring glycolysis and mitochondrial respiration in mouse splenocytes, and T and B lymphocytes.

Lymphocytes respond to a variety of stimuli by activating intracellular signaling pathways, which in turn leads to rapid cellular proliferation, migration ...

Legionella pneumophila Outer Membrane Vesicles: Isolation and Analysis of Their Pro-inflammatory Potential on Macrophages

Bacteria are able to secrete a variety of molecules via various secretory systems. Besides the secretion of molecules into the extracellular space or directly into another cell, Gram-negative bacteria can also form outer membrane vesicles (OMVs). These membrane vesicles can deliver their cargo over long distances, and the cargo is protected from degradation by proteases and nucleases. 
Legionella pneumophila (L. pneumophila) is an intracellular, Gram-negative pathogen that causes a severe form of pneumonia. In humans, it infects alveolar macrophages, where it blocks lysosomal degradation and forms a specialized replication vacuole. Moreover, L. pneumophila produces OMVs under various growth conditions. To understand the role of OMVs in the infection process of human macrophages, we set up a protocol to purify bacterial membrane vesicles from liquid culture. The method is based on differential ultracentrifugation. The enriched OMVs were subsequently analyzed with regard to their protein and lipopolysaccharide (LPS) amount and were then used for the treatment of a human monocytic cell line or murine bone marrow-derived macrophages. The pro-inflammatory responses of those cells were analyzed by enzyme-linked immunosorbent assay. Furthermore, alterations in a subsequent infection were analyzed. To this end, the bacterial replication of L. pneumophila in macrophages was studied by colony-forming unit assays. 
Here, we describe a detailed protocol for the purification of L. pneumophila OMVs from liquid culture by ultracentrifugation and for the downstream analysis of their pro-inflammatory potential on macrophages.

Legionella pneumophila Outer Membrane Vesicles: Isolation and Analysis of Their Pro-inflammatory Potential on Macrophages

Here, we describe the purification of Legionella pneumophila (L. pneumophila) outer membrane vesicles (OMVs) from liquid cultures. These purified vesicles are then used for the treatment of macrophages to analyze their pro-inflammatory potential.

Bacteria are able to secrete a variety of molecules via various secretory systems. Besides the secretion of molecules into the extracellular space or ...

Precision-cut Mouse Lung Slices to Visualize Live Pulmonary Dendritic Cells

Inhalation of allergens and pathogens elicits multiple changes in a variety of immune cell types in the lung. Flow cytometry is a powerful technique for quantitative analysis of cell surface proteins on immune cells, but it provides no information on the localization and migration patterns of these cells within the lung. Similarly,&#160;chemotaxis assays can be performed to study the potential of cells to respond to chemotactic factors in vitro, but these assays do not reproduce the complex environment of the intact lung. In contrast to these aforementioned techniques, the location of individual cell types within the lung can be readily visualized by generating Precision-cut Lung Slices (PCLS), staining them with commercially available, fluorescently tagged antibodies, and visualizing the sections by confocal microscopy. PCLS can be used for both live and fixed lung tissue, and the slices can encompass areas as large as a cross section of an entire lobe. We have used this protocol to successfully visualize the location of a wide variety of cell types in the lung, including distinct types of dendritic cells, macrophages, neutrophils, T cells and B cells, as well as structural cells such as lymphatic, endothelial, and epithelial cells. The ability to visualize cellular interactions, such as those between dendritic cells and T cells, in live, three-dimensional lung tissue, can reveal how cells move within the lung and interact with one another at steady state and during inflammation. Thus, when used in combination with other procedures, such as flow cytometry and quantitative PCR, PCLS can contribute to a comprehensive understanding of cellular events that underlie allergic and inflammatory diseases of the lung.

We describe a method for generating Precision-cut Lung Slices (PCLS) and immunostaining them to visualize the localization of various immune cell types in the lung. Our protocol can be extended to visualize the location and function of many different cell types under a variety of conditions.

Inhalation of allergens and pathogens elicits multiple changes in a variety of immune cell types in the lung. Flow cytometry is a powerful technique for ...

The WinCF Model - An Inexpensive and Tractable Microcosm of a Mucus Plugged Bronchiole to Study the Microbiology of Lung Infections

Many chronic airway diseases result in mucus plugging of the airways. Lungs of an individual with cystic fibrosis are an exemplary case where their mucus-plugged bronchioles create a favorable habitat for microbial colonization. Various pathogens thrive in this environment interacting with each other and driving many of the symptoms associated with CF disease. Like any microbial community, the chemical conditions of their habitat have a significant impact on the community structure and dynamics. For example, different microorganisms thrive in differing levels of oxygen or other solute concentrations. This is also true in the CF lung, where oxygen concentrations are believed to drive community physiology and structure. The methods described here are designed to mimic the lung environment and grow pathogens in a manner more similar to that from which they cause disease. Manipulation of the chemical surroundings of these microbes is then used to study how the chemistry of lung infections governs its microbial ecology. The method, called the WinCF system, is based on artificial sputum medium and narrow capillary tubes meant to provide an oxygen gradient similar to that which exists in mucus-plugged bronchioles. Manipulating chemical conditions, such as the media pH of the sputum or antibiotics pressure, allows for visualization of the microbiological differences in those samples using colored indicators, watching for gas or biofilm production, or extracting and sequencing the nucleic acid contents of each sample.

The mucus plugged airways of cystic fibrosis (CF) patients are an ideal environment for microbial pathogens to thrive. The manuscript describes a novel method for studying the CF lung microbiome in an environment that mimics where they cause disease and how alterations of chemical conditions can drive microbial dynamics.

Many chronic airway diseases result in mucus plugging of the airways. Lungs of an individual with cystic fibrosis are an exemplary case where their ...

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.