Name: a method for generating pulmonary neutrophilia using aerosolized lipopolysaccharide
Uploaded: 2014-12-15T13:00:00
Description: Watch this Scientific Journal Video about A Method for Generating Pulmonary Neutrophilia Using Aerosolized Lipopolysaccharide at JoVE.com

We would like to thank Kerstin Thim (AstraZeneca, Lund, Sweden), Benita Dahlberg and Dr. Anders Eklund (Karolinska Institutet, Stockholm, Sweden) as well as Dr. Martin Stampfli (McMaster University, Hamilton, ON, Canada) for skillful assistance and expert advice.

The animal studies were approved by the Northern Stockholm animal welfare ethics committee. The experimental procedures were performed in compliance with Swedish law.
1. Generating an LPS Aerosol
<ol>
	<li>Dissolve 0.5 g purified P. aeruginosa LPS in 50 ml sterile saline with gentle agitation and verify dissolution. Dilute 1 ml dissolved LPS in 9 ml sterile saline, to a final concentration of 1 mg/ml. Protect from light with aluminum foil and store at -20 &#176;C.</li>
	<li>Thaw sol...

<ol>
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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review:+Lipopolysaccharide+biosynthesis+in+Pseudomonas+aeruginosa.">Review: Lipopolysaccharide biosynthesis in Pseudomonas aeruginosa.</a> Innate Immun. 15, 261-312 (2009).</li><li>Grommes, J., Soehnlein, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+neutrophils+to+acute+lung+injury.">Contribution of neutrophils to acute lung injury.</a> Mol Med. 17, 293-307 (2011).</li><li>Pesci, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammatory+cells+and+mediators+in+bronchial+lavage+of+patients+with+chronic+obstructive+pulmonary+disease.">Inflammatory cells and mediators in bronchial lavage of patients with chronic obstructive pulmonary disease.</a> Eur Respir J. 12, 380-386 (1998).</li><li>Hoogerwerf, J. 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L., Zarkower, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+humoral+responses+to+Escherichia+coli+and+sheep+red+blood+cell+antigens+introduced+via+the+respiratory+tract.">Comparative humoral responses to Escherichia coli and sheep red blood cell antigens introduced via the respiratory tract.</a> Exp Lung Res. 13, 97-112 (1987).</li><li>Asti, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipopolysaccharide-induced+lung+injury+in+mice.+I.+Concomitant+evaluation+of+inflammatory+cells+and+haemorrhagic+lung+damage.">Lipopolysaccharide-induced lung injury in mice. I. Concomitant evaluation of inflammatory cells and haemorrhagic lung damage.</a> Pulm Pharmacol Ther. 13, 61-69 (2000).</li><li>Brand, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Total+deposition+of+therapeutic+particles+during+spontaneous+and+controlled+inhalations.">Total deposition of therapeutic particles during spontaneous and controlled inhalations.</a> Journal of pharmaceutical sciences. 89, 724-731 (2000).</li><li>Liu, F., Li, W., Pauluhn, J., Trubel, H., Wang, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipopolysaccharide-induced+acute+lung+injury+in+rats:+comparative+assessment+of+intratracheal+instillation+and+aerosol+inhalation.">Lipopolysaccharide-induced acute lung injury in rats: comparative assessment of intratracheal instillation and aerosol inhalation.</a> Toxicology. 304, 158-166 (2013).</li><li>Skerrett, S. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+the+type+1+TNF+receptor+in+lung+inflammation+after+inhalation+of+endotoxin+or+Pseudomonas+aeruginosa.">Role of the type 1 TNF receptor in lung inflammation after inhalation of endotoxin or Pseudomonas aeruginosa.</a> American Journal of Physiology - Lung Cellular and Molecular Physiology. 276, L715-L727 (1999).</li><li>Roos, A. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lung+epithelial-C/EBPbeta+contributes+to+LPS-induced+inflammation+and+its+suppression+by+formoterol.">Lung epithelial-C/EBPbeta contributes to LPS-induced inflammation and its suppression by formoterol.</a> Biochem Biophys Res Commun. 423, 134-139 (2012).</li><li>Didon, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lung+epithelial+CCAAT/enhancer-binding+protein-beta+is+necessary+for+the+integrity+of+inflammatory+responses+to+cigarette+smoke.">Lung epithelial CCAAT/enhancer-binding protein-beta is necessary for the integrity of inflammatory responses to cigarette smoke.</a> Am J Respir Crit Care Med. 184, 233-242 (2011).</li><li>Silverpil, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Negative+feedback+on+IL-23+exerted+by+IL-17A+during+pulmonary+inflammation.">Negative feedback on IL-23 exerted by IL-17A during pulmonary inflammation.</a> Innate Immunity. 19, 479-492 (2013).</li><li>Mercer, P. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteinase-Activated+Receptor-1,+CCL2+and+CCL7+Regulate+Acute+Neutrophilic+Lung+Inflammation.">Proteinase-Activated Receptor-1, CCL2 and CCL7 Regulate Acute Neutrophilic Lung Inflammation.</a> American Journal of Respiratory Cell and Molecular Biology. , (2013).</li><li>Korkmaz, B., Horwitz, M. S., Jenne, D. E., Gauthier, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Neutrophil Elastase, Proteinase 3, and Cathepsin G as Therapeutic Targets in Human Diseases. Pharmacological Reviews. 62, 726-759 (2010).</li><li>Bafadhel, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+Exacerbations+of+COPD:+Identification+of+Biological+Clusters+and+Their+Biomarkers.">Acute Exacerbations of COPD: Identification of Biological Clusters and Their Biomarkers.</a> Am. J. Respir. Crit. Care Med. , 201104-200597 (2011).</li><li>Hurst, J. R., Perera, W. R., Wilkinson, T. M. A., Donaldson, G. C., Donaldson, G. C., Wedzicha, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systemic+and+Upper+and+Lower+Airway+Inflammation+at+Exacerbation+of+Chronic+Obstructive+Pulmonary.">Systemic and Upper and Lower Airway Inflammation at Exacerbation of Chronic Obstructive Pulmonary.</a> Am. J. Respir. Crit. Care Med. 173, 71-78 (2006).</li><li>Rabe, K. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+Strategy+for+the+Diagnosis,+Management,+and+Prevention+of+Chronic+Obstructive+Pulmonary+Disease:+GOLD+Executive+Summary.">Global Strategy for the Diagnosis, Management, and Prevention of Chronic Obstructive Pulmonary Disease: GOLD Executive Summary.</a> Am. J. Respir. Crit. Care Med. 176, 532-555 (2007).</li></ol>

Aerosolized LPS generates an inflammatory response in the airways, characterized by neutrophils in the epithelial submucosa, spaces surrounding the conducting airways, as well as the alveolar spaces. This is, together with the increased total protein content in BALF, indicative of plasma leakage, representative of the pathology of acute lung injury. As LPS induces a sterile inflammation, the reaction is independent of the adaptive immune response, and there are limitations to the relevance to bacterial infections. The te...

Lipopolysaccharide (LPS) is a cell wall component of gram negative bacteria1. Challenge to LPS is a well-documented model of acute lung injury, a syndrome characterized by acute neutrophilic inflammation and edema2. In addition, pulmonary neutrophilia is also a hallmark of chronic obstructive pulmonary disease (COPD)3, and LPS challenge in humans has been used to model COPD exacerbations4. Thus, experimental models of LPS exposure are clinically relevant and valuable tools to understand human pathology.
The objective of the pulmonary delivery of aerosolized LPS described here is to generate a neutrophilic inflammatory response in the conducting and respiratory airways, without systemic involvement. Several techniques of LPS challenge have been described previously. Intra-venous injection of LPS is the most commonly used route of administration. Although this technique is easily accessible, the primary damage is to the endothelium, with secondary destruction of the pulmonary epithelium following neutrophil migration to the lung. Intra-venous administration also induces systemic inflammation2, which may complicate the clinical picture in animal models. Systemic inflammation is in contrast not observed with intra-tracheal administration. This technique, however, is labor intensive and requires anesthetics as well as considerable training5,6 . Furthermore, pulmonary deposition by this route of administration is dependent on breathing7. Thus, pulmonary deposition is affected by the depth of anesthesia needed for the intra tracheal administration and variable deposition in the airways may be observed. In contrast, pulmonary delivery with aerosolized LPS requires minimal training, and can easily be accomplished on a large number of animals with little or no variation between individuals5,8 . A recent study confirms that aerosol delivery is superior to the intra-tracheal route with regard to deposition, and that more relevant doses of LPS induce neutrophilic inflammation with this model8.
Previous studies have demonstrated that challenge to aerosolized Psuedomonas aeruginosa LPS generates a marked inflammatory response in the airway lumen and lung parenchyma, including the alveolar spaces9,10. The inflammation is characterized by a predominance of neutrophils and presence of pulmonary edema, and can thus be used to address pathogenesis of acute lung injury and gain further knowledge of the mechanisms contributing to disease pathology.

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			<td height="22" style="height:22px;width:307px;">Name of the material/equipment&#160;</td>
			<td style="width:244px;">Company</td>
			<td style="width:145px;">Catalog number</td>
			<td style="width:235px;">Comments/Description</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:307px;">Purified Pseudomonas aeruginosa LPS&#160;</td>
			<td style="width:244px;">Sigma-Aldrich</td>
			<td style="width:145px;"></td>
			<td style="width:235px;">Harmful. Recomended purification. LPS purified from other bactria may be used.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Pari LC sprint star nebulizer</td>
			<td style="width:244px;">PARI Respiratory Equipment Inc.&#160;</td>
			<td style="width:145px;">023G1250</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:307px;">TSI mass flowmeter 4040</td>
			<td style="width:244px;">TSI</td>
			<td style="width:145px;">4040</td>
			<td style="width:235px;">Alternative product from supplier may be used.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Saint-Gobain 15.9 mm Tygon tube</td>
			<td style="width:244px;">Sigma-Aldrich</td>
			<td style="width:145px;">Z685704</td>
			<td style="width:235px;">Recomended brand.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:307px;">Plexiglas boxes with removable lids</td>
			<td style="width:244px;">Custom built</td>
			<td style="width:145px;">N/A</td>
			<td style="width:235px;">150 x 163 x 205 mm (a 2 mm hole on the side).&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">3M Half Facepiece Reusable Respirator</td>
			<td style="width:244px;">3M</td>
			<td style="width:145px;">7503</td>
			<td style="width:235px;">Recomended brand.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">3M Advanced Particulate Filters (P100)&#160;</td>
			<td style="width:244px;">3M</td>
			<td style="width:145px;">2291</td>
			<td style="width:235px;">Recomended brand.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Sissors</td>
			<td style="width:244px;">VWR</td>
			<td style="width:145px;">233-1104</td>
			<td style="width:235px;">Preferred scissors may be used.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Forceps&#160;</td>
			<td style="width:244px;">VWR</td>
			<td style="width:145px;">232-1313</td>
			<td style="width:235px;">Preferred forceps may be used.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Intramedic PE50 polyethylene tube</td>
			<td style="width:244px;">BD</td>
			<td style="width:145px;">427411</td>
			<td style="width:235px;">Recomended brand.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Ethicon 2-0 Perma-hand silk tread&#160;</td>
			<td style="width:244px;">VWR</td>
			<td style="width:145px;">95056-992</td>
			<td style="width:235px;">Recomended brand.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">26 &#189;&#160; gage needle&#160;</td>
			<td style="width:244px;"></td>
			<td style="width:145px;"></td>
			<td style="width:235px;">Alternative suppliers exist.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">1 mL BD slip-tip syringe, non-sterile</td>
			<td style="width:244px;">BD</td>
			<td style="width:145px;"><a href="http://catalog.bd.com/bdCat/viewProduct.doCustomer?productNumber=301025">301025</a></td>
			<td style="width:235px;">Alternative suppliers exist.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:307px;">60 mL BD Luer-Lok syringe, non-sterile, polypropolene&#160;</td>
			<td style="width:244px;">BD</td>
			<td style="width:145px;">301035</td>
			<td style="width:235px;">Alternative suppliers exist.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Fluka Hematoxylin-Eosin</td>
			<td style="width:244px;">Sigma-Aldrich</td>
			<td style="width:145px;">3972</td>
			<td style="width:235px;">Alternative suppliers exist.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">T&#252;rk&#39;s solution</td>
			<td style="width:244px;">Merck Millipore</td>
			<td style="width:145px;">109277</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Table top centrifuge</td>
			<td style="width:244px;"></td>
			<td style="width:145px;"></td>
			<td style="width:235px;">Alternative manufacturers exist.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Cytospin 4 cytocentrifuge</td>
			<td style="width:244px;">Thermo Scientific</td>
			<td style="width:145px;">A78300003</td>
			<td style="width:235px;">Alternative centrifuge can be used.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">HEMA-3 stat pack</td>
			<td style="width:244px;">Fisher Scientific</td>
			<td style="width:145px;">23-123-869</td>
			<td style="width:235px;">Alternative staining kits exists.</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:307px;">Formalin solution, neutral buffered, 10%</td>
			<td style="width:244px;">Sigma-Aldrich</td>
			<td style="width:145px;">HT501128&#160;</td>
			<td style="width:235px;">Alternative suppliers exist.</td>
		</tr>
	</tbody>
</table>

a method for generating pulmonary neutrophilia using aerosolized lipopolysaccharide

Acute lung injury (ALI) is a severe disease characterized by alveolar neutrophilia, with limited treatment options and high mortality. Experimental models of ALI are key in enhancing our understanding of disease pathogenesis. Lipopolysaccharide (LPS) derived from gram positive bacteria induces neutrophilic inflammation in the airways and lung parenchyma of mice. Efficient pulmonary delivery of compounds such as LPS is, however, difficult to achieve. In the approach described here, pulmonary delivery in mice is achieved by challenge to aerosolized Pseudomonas aeruginosa LPS. Dissolved LPS was aerosolized by a nebulizer connected to compressed air. Mice were exposed to a continuous flow of LPS aerosol in a Plexiglas box for 10 min, followed by 2 min conditioning after the aerosol was discontinued. Tracheal intubation and subsequent bronchoalveolar lavage, followed by formalin perfusion was next performed, which allows for characterization of the sterile pulmonary inflammation. Aerosolized LPS generates a pulmonary inflammation characterized by alveolar neutrophilia, detected in bronchoalveolar lavage and by histological assessment. This technique can be set up at a small cost with few appliances, and requires minimal training and expertise. The exposure system can thus be routinely performed at any laboratory, with the potential to enhance our understanding of lung pathology.

Challenge to aerosolized P. aeruginosa LPS usually yields a marked inflammatory response in the airway lumen and alveolar space, characterized by a predominance of neutrophils at both early and late time points.
Aerosolized LPS induces pulmonary neutrophilia
C57BL/6by and BALB/c mice were exposed to aerosolized P. aeruginosa LPS or vehicle alone and neutrophils were enumerated in BALF. The total cell number in BALF of C57BL/6by mice exposed to an aero...

Watch this Scientific Journal Video about A Method for Generating Pulmonary Neutrophilia Using Aerosolized Lipopolysaccharide at JoVE.com

A Method for Generating Pulmonary Neutrophilia Using Aerosolized Lipopolysaccharide

We describe a method for inducing neutrophilic pulmonary inflammation by challenge to aerosolized lipopolysaccharide by nebulization, to model acute lung injury. In addition, basic surgical techniques for lung isolation, tracheal intubation and bronchoalveolar lavage are also described.

Acute lung injury (ALI) is a severe disease characterized by alveolar neutrophilia, with limited treatment options and high mortality. Experimental models ...

a-method-for-generating-pulmonary-neutrophilia-using-aerosolized

Research

JoVE Journal

Immunology and Infection

A Method for Labeling Vasculature in Embryonic Mice

The establishment of a functional blood vessel network is an essential part of organogenesis, and is required for optimal organ function. For example, in the thymus proper vasculature formation and patterning is essential for thymocyte entry into the organ and mature T-cell exit to the periphery. The spatial arrangement of blood vessels in the thymus is dependent upon signals from the local microenvironment, namely thymic epithelial cells (TEC). Several recent reports suggest that disruption of these signals results in thymus blood vessel defects 1,2. Previous studies have described techniques used to label the neonatal and adult thymus vasculature 1,2. We demonstrate here a technique for labeling blood vessels in the embryonic thymus. This method combines the use of FITC-dextran or Griffonia (Bandeiraea) Simplicifolia Lectin I (GSL 1 - isolectin B4) facial vein injections and CD31 antibody staining to identify thymus vascular structures and PDGFR-&beta; to label thymic perivascular mesenchyme 3-5. The option of using cryosections or vibratome sections is also provided. This protocol can be used to identify thymus vascular defects, which is critical for defining the roles of TEC-derived molecules in thymus blood vessel formation. As the method labels the entire vasculature, it can also be used to analyze the vascular networks in multiple organs and tissues throughout the embryo including skin and heart 6-10.

This article describes a method for labeling embryonic skin and thymus blood vessels.

The establishment of a functional blood vessel network is an essential part of organogenesis, and is required for optimal organ function. For example, in ...

Diagnosing Pulmonary Tuberculosis with the Xpert MTB/RIF Test

Tuberculosis (TB) due to Mycobacterium tuberculosis (MTB) remains a major public health issue: the infection affects up to one third of the world population1, and almost two million people are killed by TB each year.2 Universal access to high-quality, patient-centered treatment for all TB patients is emphasized by WHO's Stop TB Strategy.3 The rapid detection of MTB in respiratory specimens and drug therapy based on reliable drug resistance testing results are a prerequisite for the successful implementation of this strategy. However, in many areas of the world, TB diagnosis still relies on insensitive, poorly standardized sputum microscopy methods. Ineffective TB detection and the emergence and transmission of drug-resistant MTB strains increasingly jeopardize global TB control activities.2
Effective diagnosis of pulmonary TB requires the availability - on a global scale - of standardized, easy-to-use, and robust diagnostic tools that would allow the direct detection of both the MTB complex and resistance to key antibiotics, such as rifampicin (RIF). The latter result can serve as marker for multidrug-resistant MTB (MDR TB) and has been reported in &gt; 95% of the MDR-TB isolates.4, 5 The rapid availability of reliable test results is likely to directly translate into sound patient management decisions that, ultimately, will cure the individual patient and break the chain of TB transmission in the community.2
Cepheid's (Sunnyvale, CA, U.S.A.) Xpert MTB/RIF assay6, 7 meets the demands outlined above in a remarkable manner. It is a nucleic-acids amplification test for 1) the detection of MTB complex DNA in sputum or concentrated sputum sediments; and 2) the detection of RIF resistance-associated mutations of the rpoB gene.8 It is designed for use with Cepheid's GeneXpert Dx System that integrates and automates sample processing, nucleic acid amplification, and detection of the target sequences using real-time PCR and reverse transcriptase PCR. The system consists of an instrument, personal computer, barcode scanner, and preloaded software for running tests and viewing the results.9 It employs single-use disposable Xpert MTB/RIF cartridges that hold PCR reagents and host the PCR process. Because the cartridges are self-contained, cross-contamination between samples is eliminated.6 Current nucleic acid amplification methods used to detect MTB are complex, labor-intensive, and technically demanding. The Xpert MTB/RIF assay has the potential to bring standardized, sensitive and very specific diagnostic testing for both TB and drug resistance to universal-access point-of-care settings3, provided that they will be able to afford it. In order to facilitate access, the Foundation for Innovative New Diagnostics (FIND) has negotiated significant price reductions. Current FIND-negotiated prices, along with the list of countries eligible for the discounts, are available on the web.10

The Xpert MTB/RIF test integrates sample decontamination, hands-free operation, on-board sample processing, and ultra-sensitive hemi-nested PCR for the simultaneous detection of Mycobacterium tuberculosis and rifampicin resistance, either in expectorated sputum or concentrated sputum sediments, in approximately two hours. Testing is standardized and requires only moderate laboratory infrastructure and training.

Tuberculosis (TB) due to Mycobacterium tuberculosis (MTB) remains a major public health issue: the infection affects up to one third of the world ...

Peptide:MHC Tetramer-based Enrichment of Epitope-specific T cells

A basic necessity for researchers studying adaptive immunity with in vivo experimental models is an ability to identify T cells based on their T cell antigen receptor (TCR) specificity. Many indirect methods are available in which a bulk population of T cells is stimulated in vitro with a specific antigen and epitope-specific T cells are identified through the measurement of a functional response such as proliferation, cytokine production, or expression of activation markers1. However, these methods only identify epitope-specific T cells exhibiting one of many possible functions, and they are not sensitive enough to detect epitope-specific T cells at naive precursor frequencies. A popular alternative is the TCR transgenic adoptive transfer model, in which monoclonal T cells from a TCR transgenic mouse are seeded into histocompatible hosts to create a large precursor population of epitope-specific T cells that can be easily tracked with the use of a congenic marker antibody2,3. While powerful, this method suffers from experimental artifacts associated with the unphysiological frequency of T cells with specificity for a single epitope4,5. Moreover, this system cannot be used to investigate the functional heterogeneity of epitope-specific T cell clones within a polyclonal population.
	The ideal way to study adaptive immunity should involve the direct detection of epitope-specific T cells from the endogenous T cell repertoire using a method that distinguishes TCR specificity solely by its binding to cognate peptide:MHC (pMHC) complexes. The use of pMHC tetramers and flow cytometry accomplishes this6, but is limited to the detection of high frequency populations of epitope-specific T cells only found following antigen-induced clonal expansion. In this protocol, we describe a method that coordinates the use of pMHC tetramers and magnetic cell enrichment technology to enable detection of extremely low frequency epitope-specific T cells from mouse lymphoid tissues3,7. With this technique, one can comprehensively track entire epitope-specific populations of endogenous T cells in mice at all stages of the immune response.

This protocol describes the use of peptide:MHC tetramers and magnetic microbeads to isolate low frequency populations of epitope-specific T cells and analyze them by flow cytometry. This method enables the direct study of endogenous T cell populations of interest from in vivo experimental systems.

A  basic necessity for researchers studying adaptive immunity with in vivo experimental models is an  ability to identify T cells based on their T cell ...

Cecal Ligation and Puncture-induced Sepsis as a Model To Study Autophagy in Mice

Experimental sepsis can be induced in mice using the cecal ligation and puncture (CLP) method, which causes polymicrobial sepsis. Here, a protocol is provided to induce sepsis of varying severity in mice using the CLP technique. Autophagy is a fundamental tissue response to stress and pathogen invasion. Two current protocols to assess autophagy in vivo in the context of experimental sepsis are also presented here. (I) Transgenic mice expressing green fluorescence protein (GFP)-LC3 fusion protein are subjected to CLP. Localized enhancement of GFP signal (puncta), as assayed either by immunohistochemical or confocal assays, can be used to detect enhanced autophagosome formation and, thus, altered activation of the autophagy pathway. (II) Enhanced autophagic vacuole (autophagosome) formation per unit tissue area (as a marker of autophagy stimulation) can be quantified using electron microscopy. The study of autophagic responses to sepsis is a critical component of understanding the mechanisms by which tissues respond to infection. Research findings in this area may ultimately contribute towards understanding the pathogenesis of sepsis, which represents a major problem in critical care medicine.

Experimental sepsis can be induced in mice using the cecal ligation and puncture (CLP) method. Current protocols to assess autophagy in vivo in the context of CLP-induced sepsis are presented here: A protocol for measuring autophagy using (GFP)-LC3 mice, and a protocol for measuring autophagosome formation by electron microscopy.

Experimental sepsis can be induced in mice using the cecal ligation and puncture (CLP) method, which causes polymicrobial sepsis. Here, a protocol is ...

Two-photon Intravital Imaging of Leukocytes During the Immune Response in Lipopolysaccharide-treated Mouse Liver

Sepsis is a type of severe infection that can cause organ failure and tissue damage. Although the mortality and morbidity rates associated with sepsis are extremely high, no direct treatment or organ-related mechanism has been examined in detail in real time. The liver is the key organ that manages toxins and infections in the human body. Herein, we aimed to perform intravital imaging of mouse liver after induction of endotoxemia in order to track the motility of immune cells, such as neutrophils and liver capsular macrophages (LCMs). Accordingly, we designed a novel surgical method for exposure of the liver with minimally invasive surgery. Mice were intraperitoneally injected with lipopolysaccharide (LPS), a common endotoxin. Using our novel surgical approach for exposure and intravital imaging of the mouse liver, we found that neutrophil recruitment in LPS-treated LysM-green fluorescent protein (GFP) mouse liver was increased compared with that in phosphate-buffered saline-treated liver. After LPS treatment, the number of neutrophils increased significantly with time. Additionally, using CX3Cr1-GFP mice, we successfully visualized liver resident macrophages called LCMs. Therefore, to investigate the efficacy of new reagents to control immune mobility in vivo, determining the motility and morphology of neutrophils and LCMs in the liver may allow us to identify therapeutic effect in organ failure and tissue damage caused by leukocytes activation in sepsis.

We established a novel surgical protocol for two-photon imaging of live mice liver with minimal invasion. With this technique, we identified the detailed structure of the liver during lipopolysaccharide-induced endotoxemia. We anticipate that this method may be utilized to determine the effectiveness of various reagents treatment to hepatic leukocyte migration.

Sepsis is a type of severe infection that can cause organ failure and tissue damage. Although the mortality and morbidity rates associated with sepsis are ...

Generating Recombinant Avian Herpesvirus Vectors with CRISPR/Cas9 Gene Editing

Herpesvirus of turkeys (HVT) is an ideal viral vector for the generation of recombinant vaccines against a number of avian diseases, such as avian influenza (AI), Newcastle disease (ND), and infectious bursal disease (IBD), using bacterial artificial chromosome (BAC) mutagenesis or conventional recombination methods. The clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 system has been successfully used in many settings for gene editing, including the manipulation of several large DNA virus genomes. We have developed a rapid and efficient CRISPR/Cas9-mediated genome editing pipeline to generate recombinant HVT. To maximize the potential use of this method, we present here detailed information about the methodology of generating recombinant HVT expressing the VP2 protein of IBDV. The VP2 expression cassette is inserted into the HVT genome via an NHEJ (nonhomologous end-joining)-dependent repair pathway. A green fluorescence protein (GFP) expression cassette is first attached to the insert for easy visualization and then removed via the Cre-LoxP system. This approach offers an efficient way to introduce other viral antigens into the HVT genome for the rapid development of recombinant vaccines.

Herpesvirus of turkeys (HVT) is widely used as a vector platform for the generation of recombinant vaccines against a number of avian diseases. This article describes a simple and rapid approach for the generation of recombinant HVT-vectored vaccines using an integrated NHEJ-CRISPR/Cas9 and Cre-Lox system.

Herpesvirus of turkeys (HVT) is an ideal viral vector for the generation of recombinant vaccines against a number of avian diseases, such as avian ...

Co-immunoprecipitation Assay for Studying Functional Interactions Between Receptors and Enzymes

Receptor-associated enzymes are the major mediators of cellular activation. These enzymes are regulated, at least in part, by physical interactions with cytoplasmic tails of the receptors. The interactions often occur through specific protein domains and result in activation of the enzymes. There are several methods to study interactions between proteins. While co-immunoprecipitation is commonly used to study domains that are required for protein-protein interactions, there are no assays that document the contribution of specific domains to activity of the recruited enzymes at the same time. Accordingly, the method described here combines co-immunoprecipitation and an on-bead enzymatic activity assay for simultaneous evaluation of interactions between proteins and associated enzymatic activation. The goal of this protocol is to identify the domains that are critical for physical interactions between a protein and enzyme and the domains that are obligatory for complete activation of the enzyme. The importance of this assay is demonstrated, as certain receptor protein domains contribute to the binding of the enzyme to the cytoplasmic tail of the receptor, while other domains are necessary to regulate the function of the same enzyme.

Here, we present a protocol for co-immunoprecipitation and an on-bead enzymatic activity assay to simultaneously study the contribution of specific protein domains of plasma membrane receptors to both enzyme recruitment and enzyme activity.

Receptor-associated enzymes are the major mediators of cellular activation. These enzymes are regulated, at least in part, by physical interactions with ...

Novel Protocol for Generating Physiologic Immunogenic Dendritic Cells

Extracorporeal photochemotherapy (ECP) is a widely used cancer immunotherapy for cutaneous T cell lymphoma (CTCL), operative in over 350 university centers worldwide. While ECP&#8217;s clinical efficacy and exemplary safety profile have driven its widespread use, elucidation of the underlying mechanisms has remained a challenge, partly owing to lack of a laboratory ECP model. To overcome this obstacle and create a simple, user-friendly platform for ECP research, we developed a scaled-down version of the clinical ECP leukocyte-processing device, suitable for work with both mouse models, and small human blood samples. This device is termed the Transimmunization (TI) chamber, or plate. In a series of landmark experiments, the miniaturized device was used to produce a cellular vaccine that regularly initiated therapeutic anti-cancer immunity in several syngeneic mouse tumor models. By removing individual factors from the experimental system and ascertaining their contribution to the in vivo anti-tumor response, we then elucidated key mechanistic drivers of ECP immunizing potential. Collectively, our results revealed that anti-tumor effects of ECP are initiated by dendritic cells (DC), physiologically generated through blood monocyte interaction with platelets in the TI plate, and loaded with antigens from tumor cells whose apoptotic cell death is finely titrated by exposure to the photoactivatable DNA cross-linking agent 8-methoxypsoralen and UVA light (8-MOPA). When returned to the mouse, this cellular vaccine leads to specific and transferable anti-tumor T cell immunity. We verified that the TI chamber is also suitable for human blood processing, producing human DCs fully comparable in activation state and profile to those derived from the clinical ECP chamber. The protocols presented here are intended for ECP studies in mouse and man, controlled generation of apoptotic tumor cells with 8-MOPA, and rapid production of physiologic human and mouse monocyte-derived DCs for a variety of applications.

The transimmunization (TI) device or plate and related protocols have been developed to replicate the key features of extracorporeal photochemotherapy (ECP), in an experimental setting, allowing for production of physiologically activated, tunable dendritic cells (DCs) for cancer immunotherapy.

Extracorporeal photochemotherapy (ECP) is a widely used cancer immunotherapy for cutaneous T cell lymphoma (CTCL), operative in over 350 university ...

Visualization of Bacterial Resistance using Fluorescent Antibiotic Probes

Fluorescent antibiotics are multipurpose research tools that are readily used for the study of antimicrobial resistance, due to their significant advantage over other methods. To prepare these probes, azide derivatives of antibiotics are synthesized, then coupled with alkyne-fluorophores using azide-alkyne dipolar cycloaddition by click chemistry. Following purification, the antibiotic activity of the fluorescent antibiotic is tested by minimum inhibitory concentration assessment. In order to study bacterial accumulation, either spectrophotometry or flow cytometry may be used, allowing for much simpler analysis than methods relying on radioactive antibiotic derivatives. Furthermore, confocal microscopy can be used to examine localization within the bacteria, affording valuable information about mode of action and changes that occur in resistant species. The use of fluorescent antibiotic probes in the study of antimicrobial resistance is a powerful method with much potential for future expansion.

Fluorescently tagged antibiotics are powerful tools that can be used to study multiple aspects of antimicrobial resistance. This article describes the preparation of fluorescently tagged antibiotics and their application to studying antibiotic resistance in bacteria. Probes can be used to study mechanisms of bacterial resistance (e.g., efflux) by spectrophotometry, flow cytometry, and microscopy.

Fluorescent antibiotics are multipurpose research tools that are readily used for the study of antimicrobial resistance, due to their significant ...

Cigarette Smoke Exposure in Mice using a Whole-Body Inhalation System

Close to 14% of adults in the United States were reported to smoke cigarettes in 2018. The effects of cigarette smoke (CS) on lungs and cardiovascular diseases have been widely studied, however, the impact of CS in other tissues and organs such as blood and bone marrow remain incompletely defined. Finding the appropriate system to study the effects of CS in rodents can be prohibitively expensive and require the purchase of commercially available systems. Thus, we set out to build an affordable, reliable, and versatile system to study the pathologic effects of CS in mice. This whole-body inhalation exposure system (WBIS) set-up mimics the breathing and puffing of cigarettes by alternating exposure to CS and clean air. Here we show that this do-it-yourself (DIY) system induces airway inflammation and lung emphysema in mice after 4-months of cigarette smoke exposure. The effects of whole-body inhalation (WBI) of CS on hematopoietic stem and progenitor cells (HSPCs) in the bone marrow using this apparatus are also shown.

This protocol demonstrates the study of the pathophysiologic effects of cigarette smoke (CS) with a whole-body inhalation (WBI) exposure system (WBIS) built in-house. This system can expose animals to CS under controlled repeatable conditions for research of CS-mediated effects on lung emphysema and hematopoiesis.

Close to 14% of adults in the United States were reported to smoke cigarettes in 2018. The effects of cigarette smoke (CS) on lungs and cardiovascular ...