This work was supported by the National Institutes of Health (NIH) AI091759 and the Crohn's and Colitis Foundation of America's William and Shelby Modell Family Foundation Research Award (to MN). Schistosome-infected mice were supplied by the NIAID Schistosome Resource Center (NIAID Contract N01 A130026).

1. Purifying Schistosoma mansoni Eggs
<ol>
 <li>Infect Swiss-Webster mice with schistosome cercariae, which is the infectious stage in the Schistosoma life cycle 8. Alternatively, obtain schistosome-infected mice from the NIAID Schistosomiasis Resource Center (<a href="http://www.schisto-resource.org/" target="_blank">http://www.schisto-resource.org/</a>). Leave mice for 6-7 weeks following infection, by which time-point, eggs are present in the liver and can be recovered as described below (See Fig. 1).</li>
 <li>Euthanize mice with CO2, and wet with 70% ethanol. Place mouse on back. Use blunt forceps and scissors to cut away skin and underlying peritoneum. Excise the liver, located below the ribcage and diaphragm.</li>
 <li>Finely mince livers and add 20 mL per liver of digestion mixture, composed of collagenase/dispase (0.5 mg/mL), penicillin (100 U/mL) and streptomycin (100 &mu;g/mL) in PBS. Put mixture in a 50 mL Falcon tube and incubate overnight in a 37 &deg;C shaker.</li>
 <li>Centrifuge mixture at 400 x g / 3 min / 20 &deg;C. Gently pour off excess liquid and fill tube with PBS. Centrifuge again and repeat PBS wash. Following last centrifugation, resuspend pellet in 25 mL PBS.</li>
 <li>Strain the resuspended pellet through a standard kitchen metallic strainer (use a 50 mL syringe plunger to mash the liver pieces through into a glass beaker), followed by passing the mixture through a fine sieve (use 10 mL syringe to push mixture through the fine sieve into a second glass beaker).</li>
 <li>Put strained mixture in a 50 mL Falcon tube. Centrifuge at 400 x g / 5 min / 20 &deg;C. Carefully pour out supernatant without losing pellet. Resuspend pellet in 3 mL PBS.</li>
 <li>Overlay mix gently onto a 20% Percoll, 40 mL gradient of 8 mL Percoll and 32 mL 0.25M sucrose (FW=342.3 so 8.56 g sucrose/100 mL H20).</li>
 <li>Centrifuge 10 min / 800 x g / 20 &deg;C. Pipette off and discard gelatinous top layer of liver cells. Remove egg pellet at bottom with a transfer pipette and put into a 15 mL Falcon tube.</li>
 <li>Wash pellet 3X in 10 mL PBS/1 mM EDTA/1 mM EGTA using centrifuge settings: 3 min / 30 x g / 20 &deg;C. Resuspend in 500 &mu;L PBS.</li>
 <li>Overlay mix on a new 25% Percoll, 10 mL gradient (2.5 mL Percoll and 7.5 mL 0.25M sucrose) using centrifuge settings: 10 min / 800 x g / 20 &deg;C.</li>
 <li>Wash 3X in 10 mL PBS using centrifuge settings: 3 min / 30 x g / 20 &deg;C.</li>
 <li>In the last 10 mL wash, remove 100 &mu;L to count eggs. Eggs settle quickly, so the Falcon tube must be mixed well before removing sample. Dilute the 100 &mu;L sample with 900 &mu;L PBS. 100 &mu;L of the mix should be counted as droplets on a microscope slide using a dissecting microscope (1:10 dilution). Wide-bore pipette tips should be used.</li>
 <li>Resuspend eggs at 50,000 eggs/mL in PBS. Eggs can be stored for several months at -80 &deg;C and thawed once or twice for use. Before use, inspect under a microscope to ensure that eggs are still intact (see Fig. 1).</li>
</ol>
2. Intraperitoneal Sensitization with S. mansoni Eggs: Day 0
<ol>
 <li>Prepare eggs at 5,000 eggs/100 &mu;L PBS in a 5 mL snapcap tube. For injection, estimate 50% more eggs than necessary.</li>
 <li>Load a 23 gauge3/4inch needle and a 1 mL syringe with 100 &mu;L of egg suspension per mouse. Eggs settle, so load needle with eggs immediately prior to use.</li>
 <li>Rock syringe back and forth to mix eggs prior to each injection, and inject intraperitoneally 100 &mu;L of suspension per mouse.</li>
</ol>
3. Retro-orbital Intravenous Challenge with S. mansoni Eggs: Day 14
<ol>
 <li>Prepare eggs as above at 5,000 eggs/100 &mu;L.</li>
 <li>Anesthetize mice in specialized chamber with isoflurane (4%), or intraperitoneally with xylazine (10 mg/kg) and ketamine (120 mg/kg). Depth of anesthesia is determined by pinching of the footpad and ensuring no physical response.</li>
 <li>Following loading of the 1 mL syringe and 23 gauge3/4inch needle with eggs, use forceps to bend the needle at a 90&deg; angle with the bevel facing down into the angle. Ensure that no bubbles are present.</li>
 <li>Place mouse on one side. Retract skin so eye will protrude, and inject 100 &mu;L into the vessels behind the eyeball at a 45&deg; angle to the nose.</li>
 <li>Withdraw needle and apply light pressure to control bleeding.</li>
</ol>
All of these procedures must be carried out with care, and mice must be monitored until they recover from anesthesia. These protocols were approved by the University of Pennsylvania Institutional Animal Care and Use Committee (IACUC).
4. Experimental Harvest: Day 22
<ol>
 <li>Euthanize mice with CO2.</li>
 <li>Wet mouse with 70% ethanol, and place on back.</li>
 <li>Using blunt forceps, grasp the mouse abdomen and make a small incision in the skin. Carefully remove skin with scissors and expose the rib cage and peritoneum.</li>
 <li>Cut open the peritoneum. Cut the abdominal aorta to recover the blood with a Pasteur pipette. Store on ice.</li>
 <li>Move the liver down to expose the diaphragm. Use fine scissors to cut the diaphragm. Carefully cut open the rib cage to expose the lung tissue.</li>
 <li>Recover the parathymic lymph nodes that drain the lung with fine forceps. These are tucked underneath the rib cage, on either side of the thymus 9. Store on ice in 1mL sterile media (DMEM supplemented with 10% heat-inactivated fetal calf serum, 100 U/mL penicillin, 100 &mu;g/mL streptomycin, 2 mM L-glutamine, 50 &mu;M 2-mercaptoethanol, all available from Invitrogen).</li>
 <li>To recover the BAL, and inflate the lungs for histologic analysis, intratracheal intubation must be performed. Expose the trachea by moving the salivary glands to the side, and placing fine forceps under the trachea. Using fine scissors, cut a hole in the middle of the trachea, and insert tubing into the lungs. Secure tubing by tying with surgical thread.</li>
 <li>Attach PBS-filled 1 mL syringe to the tubing and inflate lungs with 1 mL PBS. Carefully retrieve BAL wash with syringe. Store on ice.</li>
 <li>Attach 4% paraformaldehyde (PFA) in PBS-filled 1 mL syringe to the tubing and inflate the lungs.</li>
 <li>Remove the tubing, and tighten the surgical thread to prevent leaking of the PFA.</li>
 <li>Carefully dissect out lung tissue and place in a 50 mL Falcon tube with 5 mL 4%PFA.</li>
</ol>
5. Preparation of Recovered Tissues
<ol>
 <li>Serum: Following storage on ice for 30 min to 2 h for blood to clot, serum is recovered from the blood by centrifugation (13000 x g / 10 min / 4 &deg;C). Serum is stored at -20 &deg;C and can be used for cytokine ELISAs or S. mansoni egg antigen-specific IgG isotype ELISAs 6, 10.</li>
 <li>Lymph Nodes: Single cell suspensions are prepared and cell counts used to evaluate the magnitude of the immune response. To examine Th2 cell polarization, cells are re-stimulated with S. mansoni egg antigen for 72 hours. Intracellar cytokine staining can be examined by flow cytometry, and supernatants can be recovered and stored at -20 &deg;C for ELISA of Th2 cytokines (IL-4, IL-5, IL-13) 6.</li>
 <li>BAL: Cells are pelleted (400 x g / 5 min / 4 &deg;C). BAL fluid is recovered and stored at -20 &deg;C for analysis of Th2 cytokines by ELISA. BAL cells counts are used as a readout for airway inflammation. Cytocentrifuge preparations of 10,000-100,000 cells (500 rpm / 5 min / 4 &deg;C), followed by Diffquik staining will allow enumeration of inflammatory infiltrate 4, 6. Alternatively, BAL cells can be examined by flow cytometry 6.</li>
 <li>Lung tissue: Lung tissue is stored overnight at 4 &deg;C. Fixed tissue is paraffin-embedded, sectioned and mounted on slides for histological and immunofluorescence analysis.</li>
</ol>
6. Representative Results
This protocol details all the steps necessary to (i) prepare purified Schistosoma mansoni eggs for in vivo use (Fig. 1), (ii) sensitize and challenge mice with these eggs and (iii) recover organs for examination of the lung inflammatory response. This in vivo model reproducibly drives a potent Th2 lung inflammatory response. This is shown by airway inflammation and eosinophilia as visualized by BAL cell counts and cytocentrifuge preparations (Fig. 2). H&amp;E-stained lung sections can be examined for egg-induced granulomas (Fig. 3a), and immunofluorescent staining of lung sections allows visualization of alternatively activated macrophages and Th2 cytokine-induced genes such as Resistin-like molecule (RELM) &alpha; (Fig. 3b). The antigen-specific CD4 Th2 cytokine response is examined by ELISAs for IL-5, IL-4 and IL-13 of antigen-stimulated parathymic lymph node cells (Fig. 4).
<img alt="Figure 1" src="/files/ftp_upload/3905/3905fig1.jpg" /> 
Figure 1. Schistosoma mansoni egg injection model. Representative pictures of granulomatous livers, characteristic of high S.mansoni-egg burdens, and of S.mansoni eggs are shown - average size (L) 120 &mu;m by (W) 50 &mu;m.
<img alt="Figure 2" src="/files/ftp_upload/3905/3905fig2.jpg" /> 
Figure 2. S. mansoni egg injection drives airway inflammation. A. BAL cell numbers (x105) from na&iuml;ve (N) or S.mansoni (Sm) egg-injected mice. *P&lt;0.05. B. Cytocentrifuge preparation of BAL cells. Mac, MACROPHAGE; Eos, EOSINOPHIL; LYMPH, lymphocyte. Bar, 10 &mu;m.
<img alt="Figure 3" src="/files/ftp_upload/3905/3905fig3.jpg" /> 
Figure 3. Visualization of pulmonary granulomas and alternatively activated macrophages surrounding the S.mansoni egg. A. Pulmonary granuloma surrounding the Sm egg (black arrow) was visualized in H&amp;E-stained lung sections. B. Immunofluorescence staining for RELM&alpha; (green), mannose receptor (red) and DAPI (blue) reveals alternatively activated macrophages (white arrow) in the granuloma surrounding the autofluorescent Sm egg (black arrow). Bar, 50 &mu;m.
<img alt="Figure 4" src="/files/ftp_upload/3905/3905fig4.jpg" /> 
Figure 4. S.mansoni egg antigen-specific Th2 cytokine response. Draining parathymic lymph node cells from na&iuml;ve (N) or Sm egg-injected mice were stimulated with Sm egg antigen for 72 hours, followed by ELISA of supernatants for IL-4, IL-5 and IL-13. Scale, ng/mL.*P&lt;0.05.

<ol>
	<li>World Health Organization. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevention+and+control+of+schistosomiasis+and+soil-transmitted+helminthiasis:+report+of+a+WHO+expert+committee.">Prevention and control of schistosomiasis and soil-transmitted helminthiasis: report of a WHO expert committee.</a> WHO Technical Report Series 912. , (2002).</li><li>Pearce, E. J., MacDonald, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+immunobiology+of+schistosomiasis.">The immunobiology of schistosomiasis.</a> Nat. Rev. Immunol. 2, 499-511 (2002).</li><li>Sandler, N. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+gene+expression+profiles+during+acute+pathogen-induced+pulmonary+inflammation+reveal+divergent+roles+for+Th1+and+th2+responses+in+tissue+repair.">Global gene expression profiles during acute pathogen-induced pulmonary inflammation reveal divergent roles for Th1 and th2 responses in tissue repair.</a> J. Immunol. 171, 3655-3667 (2003).</li><li>Perrigoue, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IL-31-IL-31R+interactions+negatively+regulate+type+2+inflammation+in+the+lung.">IL-31-IL-31R interactions negatively regulate type 2 inflammation in the lung.</a> J. Exp. Med. 204, 481-487 (2007).</li><li>Sandor, M., Weinstock, J. V., Wynn, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Granulomas+in+schistosome+and+mycobacterial+infections:+a+model+of+local+immune+responses.">Granulomas in schistosome and mycobacterial infections: a model of local immune responses.</a> Trends Immunol. 24, 44-52 (2003).</li><li>Nair, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alternatively+activated+macrophage-derived+RELM-{alpha}+is+a+negative+regulator+of+type+2+inflammation+in+the+lung.">Alternatively activated macrophage-derived RELM-{alpha} is a negative regulator of type 2 inflammation in the lung.</a> J. Exp. Med. 206, 937-952 (2009).</li><li>Dalton, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+the+isolation+of+schistosome+eggs+and+miracidia+free+of+contaminating+host+tissues.">A method for the isolation of schistosome eggs and miracidia free of contaminating host tissues.</a> Parasitology. 115, 29-32 (1997).</li><li>Lewis, F. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-scale+laboratory+maintenance+of+Schistosoma+mansoni,+with+observations+on+three+schistosome/snail+host+combinations.">Large-scale laboratory maintenance of Schistosoma mansoni, with observations on three schistosome/snail host combinations.</a> J. Parasitol. 72, 813-829 (1986).</li><li>Tilney, N. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterns+of+lymphatic+drainage+in+the+adult+laboratory+rat.">Patterns of lymphatic drainage in the adult laboratory rat.</a> J. Anat. 109 (Pt. 3), 369-383 (1971).</li><li>Lewis, F., Coligan, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Schistosomiasis.">Schistosomiasis.</a> Current protocols in immunology. Chapter 19, Unit 19 (2001).</li><li>Kaplan, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Th2+cells+are+required+for+the+Schistosoma+mansoni+egg-induced+granulomatous+response.">Th2 cells are required for the Schistosoma mansoni egg-induced granulomatous response.</a> J. Immunol. 160, 1850-1856 (1998).</li><li>Wynn, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibrotic+disease+and+the+T(H)1/T(H)2+paradigm.">Fibrotic disease and the T(H)1/T(H)2 paradigm.</a> Nat. Rev. Immunol. 4, 583-594 (2004).</li><li>Cheever, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variation+of+hepatic+fibrosis+and+granuloma+size+among+mouse+strains+infected+with+Schistosoma+mansoni.">Variation of hepatic fibrosis and granuloma size among mouse strains infected with Schistosoma mansoni.</a> Am. J. Trop. Med. Hyg. 37, 85-97 (1987).</li></ol>

No conflicts of interest declared.

Here, a model employing Schistosoma mansoni eggs to induce type 2 lung inflammation is described. Characteristic features of this model include the potent Th2 immune response, airway inflammation and pulmonary granuloma formation. Since these parameters are dependent on CD4 Th2 cell responses 11, this is a useful model to investigate the effects of protein- specific or lineage-specific deletions on Th2 cell responses. Here the readouts would include quantification of antigen-specific Th2 cytokines, an...

<table border="1">
	<tbody>
		<tr>
			<td>Reagent</td>
			<td>Size/Description</td>
			<td>Company</td>
			<td>Catalog #</td>
		</tr>
		<tr>
			<td>Collagenase/Dispase</td>
			<td>500 mg</td>
			<td>Roche Applied Science</td>
			<td>11 097 113 001</td>
		</tr>
		<tr>
			<td>Diff Quik Hema 3 Stain</td>
			<td>Pack</td>
			<td>Fisher Scientific Co. LLC</td>
			<td>122-911</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Liquid</td>
			<td>Gibco</td>
			<td>11965</td>
		</tr>
		<tr>
			<td>Falcon 50mL</td>
			<td>Pack</td>
			<td>Falcon</td>
			<td>352070</td>
		</tr>
		<tr>
			<td>Intramedic PE Tubing</td>
			<td>0.58 mm</td>
			<td>Becton Dickinson and Co.</td>
			<td>427410</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>250 mL</td>
			<td>Abbot Animal Health</td>
			<td>52600405</td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>100 mg/mL</td>
			<td>Fort Dodge, Animal Health</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td>Mannose receptor antibody</td>
			<td>Biotin</td>
			<td>AbD Serotec</td>
			<td>MCA2235B</td>
		</tr>
		<tr>
			<td>Metallic Strainer</td>
			<td>Standard kitchen strainer</td>
			<td>Target</td>
			<td>Laurentiis</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>16%</td>
			<td>Electron Microsc. Sciences</td>
			<td>15710</td>
		</tr>
		<tr>
			<td>Penicillin/ Streptomycin</td>
			<td>100x</td>
			<td>Gibco</td>
			<td>15070</td>
		</tr>
		<tr>
			<td>Percoll</td>
			<td>1 Liter</td>
			<td>GE Healthcare Biosciences</td>
			<td>17 0891 01</td>
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		<tr>
			<td>RELM&alpha; antibody</td>
			<td>50 &mu;g</td>
			<td>Peprotech</td>
			<td>500-P214</td>
		</tr>
		<tr>
			<td>Surgical Thread</td>
			<td>2.0 USP 100 yds</td>
			<td>Surgical Specialties Corp.</td>
			<td>SP118</td>
		</tr>
		<tr>
			<td>Syringe Needle</td>
			<td>23g, 3/4 inch</td>
			<td>BD Vacutainer Lab. Med.</td>
			<td>305143</td>
		</tr>
		<tr>
			<td>USA Standard Testing Sieve</td>
			<td>150 &mu;m, 0.0059"</td>
			<td>W.S. Tyler, Inc.</td>
			<td>ASTME-11, Spec. No. 100</td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>20 mg/mL</td>
			<td>Butler</td>
			<td>N/A</td>
		</tr>
	</tbody>
</table>

using eggs from schistosoma mansoni as an in vivo model of helminth-induced lung inflammation

Schistosoma parasites are blood flukes that infect an estimated 200 million people worldwide 1. In chronic infection with Schistosoma, the severe pathology, including liver fibrosis and splenomegaly, is caused by the immune response to the parasite eggs rather than the parasite itself 2. Parasite eggs induce a Th2 response characterized by the production of IL-4, IL-5 and IL-13, the alternative activation of macrophages and the recruitment of eosinophils. Here, we describe injection of Schistosoma mansoni eggs as a model to examine parasite-specific Th2 cytokine responses in the lung and draining lymph nodes, the formation of pulmonary granulomas surrounding the egg, and airway inflammation. 
Following intraperitoneal sensitization and intravenous challenge, S. mansoni eggs are transported to the lung via the pulmonary arteries where they are trapped within the lung parenchyma by granulomas composed of lymphocytes, eosinophils and alternatively activated macrophages 3-6. Associated with granuloma formation, inflammation in the broncho-alveolar spaces, expansion of the draining lymph nodes and CD4 T cell activation can be observed. Here we detail the protocol for isolating Schistosoma mansoni eggs from infected livers (modified from 7), sensitizing and challenging mice, and recovering the organs (broncho-alveolar lavage (BAL), lung and draining lymph nodes) for analysis. We also include representative histologic and immunologic data and suggestions for additional immunologic analysis. 
Overall, this method provides an in vivo model to investigate helminth-induced immunologic responses in the lung, which is broadly applicable to the study of Th2 inflammatory diseases including helminth infection, fibrotic diseases, allergic inflammation and asthma. Advantages of this model for the study of type 2 inflammation in the lung include the reproducibility of a potent Th2 inflammatory response in the lung and draining lymph nodes, the ease of assessment of inflammation by histologic examination of the granulomas surrounding the egg, and the potential for long-term storage of the parasite eggs.

Watch this Scientific Journal Video about Using Eggs from Schistosoma mansoni as an In vivo Model of Helminth-induced Lung Inflammation at JoVE.com

Using Eggs from Schistosoma mansoni as an In vivo Model of Helminth-induced Lung Inflammation

Schistosoma mansoni eggs are potent stimulators of the T helper type 2 (Th2) immune response, characteristic of parasite infection, asthma and allergic inflammation. This protocol utilizes S. mansoni egg injection to generate a CD4 Th2 cytokine-induced inflammatory response in the lung, characterized by lung granuloma formation around the egg, eosinophilia and macrophage alternative activation.

Using Eggs from Schistosoma mansoni as an In vivo Model of Helminth-induced Lung Inflammation

Schistosoma parasites are blood flukes that infect an estimated 200 million people worldwide 1. In chronic infection with Schistosoma, the severe ...

using-eggs-from-schistosoma-mansoni-as-an-vivo-model-helminth-induced

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Immunology and Infection

13.7K Views.  University of Pennsylvania. Schistosoma mansoni eggs are potent stimulators of the T helper type 2 (Th2) immune response, characteristic of parasite infection, asthma and allergic inflammation. This protocol utilizes S. mansoni egg injection to generate a CD4 Th2 cytokine-induced inflammatory response in the lung, characterized by lung granuloma formation around the egg, eosinophilia and macrophage alternative activation.

Video: Using Eggs from Schistosoma mansoni as an In vivo Model of Helminth-induced Lung Inflammation

Cercarial Transformation and in vitro Cultivation of Schistosoma mansoni Schistosomules

Schistosome parasites are the causative agents of schistosomiasis, a chronically debilitating disease that affects over 200 million people globally and ranks second to malaria among parasitic diseases in terms of public health and socio-economic impact (1-4). Schistosome parasites are trematode worms with a complex life cycle interchanging between a parasitic life in molluscan and mammalian hosts with intervening free-swimming stages. Briefly, free-swimming cercariae infect a mammalian host by penetrating the skin with the aid of secreted proteases, during which time the cercariae lose their tails, transforming into schistosomules. The schistosomules must now evade the host immune system, develop a gut for digestion of red blood cells, and migrate though the lungs and portal circulation en route to their final destination in the hepatic portal system and eventually the mesenteric veins (for S. mansoni) where male and female worms pair and mate, producing hundreds of eggs daily. Some of the eggs are excreted from the body into fresh water, where the eggs hatch into free-swimming miracidia (5-10). The miracidia infect specific snail species and transform into mother and daughter sporocysts, which in turn, produce infective cercariae, completing the life cycle. Unfortunately, the entire schistosome life cycle cannot be cultured in vitro, but infective cercariae can be transformed into schistosomules, and the schistosomules can be cultured for weeks for the analysis of schistosome development in vitro or microarray analysis. In this protocol, we provide a visual description of cercarial transformation and in vitro culturing of schistosomules. We shed infectious cercariae from the snail host Biomphalaria glabrata and manually transform them into schistosomules by detaching their tails using an emulsifying double-ended needle. The in vitro cercarial transformation and schistosomules culture techniques described avoid the use of a mammalian host, which simplifies visualization of schistosomes and facilitates the collection of the parasite for experimental analysis. in vitro transformation and culturing techniques of schistosomes have been done for years (11, 12), but no visual protocols have been developed that are available to the entire community.

Cercarial Transformation and in vitro Cultivation of Schistosoma mansoni Schistosomules

We describe an in vitro method for culturing schistosomula of the flatworm parasite Schistosoma mansoni, via the harvesting and transformation of infective cercariae from the fresh water snail intermediate host Biomphalaria glabrata.

Schistosome parasites are the causative agents of schistosomiasis, a chronically debilitating disease that affects over 200 million people globally and ...

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological deficiencies. GVHD is caused by mature alloreactive T cells present in the bone marrow graft that are infused into the recipient and cause damage to host organs. However, in mice, T cells must be added to the bone marrow inoculum to cause GVHD. Although extensive work has been done to characterize T cell responses post transplant, bioluminescent imaging technology is a non-invasive method to monitor T cell trafficking patterns in vivo. 
Following lethal irradiation, recipient mice are transplanted with bone marrow cells and splenocytes from donor mice. T cell subsets from L2G85.B6 (transgenic mice that constitutively express luciferase) are included in the transplant. By only transplanting certain T cell subsets, one is able to track specific T cell subsets in vivo, and based on their location, develop hypotheses regarding the role of specific T cell subsets in promoting GVHD at various time points. At predetermined intervals post transplant, recipient mice are imaged using a Xenogen IVIS CCD camera. Light intensity can be quantified using Living Image software to generate a pseudo-color image based on photon intensity (red = high intensity, violet = low intensity). 
Between 4-7 days post transplant, recipient mice begin to show clinical signs of GVHD. Cooke et al.1 developed a scoring system to quantitate disease progression based on the recipient mice fur texture, skin integrity, activity, weight loss, and posture. Mice are scored daily, and euthanized when they become moribund. Recipient mice generally become moribund 20-30 days post transplant. 
Murine models are valuable tools for studying the immunology of GVHD. Selectively transplanting particular T cell subsets allows for careful identification of the roles each subset plays. Non-invasively tracking T cell responses in vivo adds another layer of value to murine GVHD models.

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Murine bone marrow transplantation is a widely used technique to study immunological mechanisms governing graft-versus-host disease in humans. The ability to monitor T cell trafficking patterns in vivo allows for detailed analysis of the development and perpetuation of T cell responses during graft-versus-host disease.

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological ...

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

Inflammation is a fundamental aspect of many human diseases. In this video report, we demonstrate non-invasive bioluminescence imaging techniques that distinguish acute and chronic inflammation in mouse models. With tissue damage or pathogen invasion, neutrophils are the first line of defense, playing a major role in mediating the acute inflammatory response. As the inflammatory reaction progresses, circulating monocytes gradually migrate into the site of injury and differentiate into mature macrophages, which mediate chronic inflammation and promote tissue repair by removing tissue debris and producing anti-inflammatory cytokines. Intraperitoneal injection of luminol (5-amino-2,3-dihydro-1,4-phthalazinedione, sodium salt) enables detection of acute inflammation largely mediated by tissue-infiltrating neutrophils. Luminol specifically reacts with the superoxide generated within the phagosomes of neutrophils since bioluminescence results from a myeloperoxidase (MPO) mediated reaction. Lucigenin (bis-N-methylacridinium nitrate) also reacts with superoxide in order to generate bioluminescence. However, lucigenin bioluminescence is independent of MPO and it solely relies on phagocyte NADPH oxidase (Phox) in macrophages during chronic inflammation. Together, luminol and lucigenin allow non-invasive visualization and longitudinal assessment of different phagocyte populations across both acute and chronic inflammatory phases. Given the important role of inflammation in a variety of human diseases, we believe this non-invasive imaging method can help investigate the differential roles of neutrophils and macrophages in a variety of pathological conditions.

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

We describe a non-invasive imaging method for distinguishing inflammatory stages. Systemic delivery of luminol reveals areas of acute inflammation dependent upon MPO activity in neutrophils. In contrast, injection of lucigenin allows for visualization of chronic inflammation dependent upon Phox activity in macrophages.

Inflammation is a fundamental aspect of many human diseases. In this video report, we demonstrate non-invasive bioluminescence imaging techniques that ...

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

Death of oligodendrocytes accompanied by destruction of neurons and axons are typical histopathological findings in cortical and subcortical grey matter lesions in inflammatory demyelinating disorders like multiple sclerosis (MS). In these disorders, mainly CD8+ T-cells of putative specificity for myelin- and oligodendrocyte-related antigens are found, so that neuronal apoptosis in grey matter lesions may be a collateral effect of these cells. Different types of animal models are established to study the underlying mechanisms of the mentioned pathophysiological processes. However, although they mimic some aspects of MS, it is impossible to dissect the exact mechanism and time course of &#8216;&#8216;collateral&#8217;&#8217; neuronal cell death. To address this course, here we show a protocol to study the mechanisms and time response of neuronal damage following an oligodendrocyte-directed CD8+ T cell attack. To target only the myelin sheath and the oligodendrocytes, in vitro activated oligodendrocyte-specific CD8+ T-cells are transferred into acutely isolated brain slices. After a defined incubation period, myelin and neuronal damage can be analysed in different regions of interest. Potential applications and limitations of this model will be discussed.

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

To address mechanisms of demyelination and neuronal apoptosis in cortical lesions of inflammatory demyelinating disorders, different animal models are used. We here describe an ex vivo approach by using oligodendrocyte-specific CD8+ T-cells on brain slices, resulting in oligodendroglial and neuronal death. Potential applications and limitations of the model are discussed.

Death of oligodendrocytes accompanied by destruction of neurons and axons are typical histopathological findings in cortical and subcortical grey matter ...

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 Method for Isolating and Expanding Invariant Natural Killer T Cells from Mouse Spleen

The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells are therefore characterized as an innate T cell population capable of activating and steering adaptive immune responses. The development of improved techniques for the culture and expansion of murine iNKT cells facilitates the study of iNKT cell biology in in vitro and in vivo model systems. Here we describe an optimized procedure for the isolation and expansion of murine splenic iNKT cells.
Spleens from C57Bl/6 mice are removed, dissected and strained and the resulting cellular suspension is layered over density gradient media. Following centrifugation, splenic mononuclear cells (MNCs) are collected and CD5-positive (CD5+) lymphocytes are enriched for using magnetic beads. iNKT cells within the CD5+ fraction are subsequently stained with &#945;GalCer-loaded CD1d tetramer and purified by fluorescence activated cell sorting (FACS). FACS sorted iNKT cells are then initially cultured in vitro using a combination of recombinant murine cytokines and plate-bound T cell receptor (TCR) stimuli before being expanded in the presence of murine recombinant IL-7. Using this technique, approximately 108 iNKT cells can be generated within 18-20 days of culture, after which they can be used for functional assays in vitro, or for in vivo transfer experiments in mice.

Here we present an adapted protocol that can be used to generate a large number of murine invariant natural killer T cells from mouse spleen. The protocol outlines an approach by which splenic iNKT cells can be enriched for, isolated and expanded in vitro using a limited number of animals and reagents.

The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells ...

Optical Screening of Novel Bacteria-specific Probes on Ex Vivo Human Lung Tissue by Confocal Laser Endomicroscopy

Improving the speed and accuracy of bacterial detection is important for patient stratification and to ensure the appropriate use of antimicrobials. To achieve this goal, the development of diagnostic techniques to recognize bacterial presence in real-time at the point-of-care is required. Optical imaging for direct identification of bacteria within the host is an attractive approach. Several attempts at chemical probe design and validation have been investigated, however none have yet been successfully translated into the clinic. Here we describe a method for ex vivo validation of bacteria-specific probes for identification of bacteria within the distal lung, imaged by fibered confocal fluorescence microscopy (FCFM). Our model used ex vivo human lung tissue and a clinically approved confocal laser endomicroscopy (CLE) platform to screen novel bacteria-specific imaging compounds, closely mimicking imaging conditions expected to be encountered with patients. Therefore, screening compounds by this technique provides confidence of potential clinical tractability.

Optical Screening of Novel Bacteria-specific Probes on Ex Vivo Human Lung Tissue by Confocal Laser Endomicroscopy

This technique describes an efficient screening process for evaluating bacteria-specific optical imaging agents within ex vivo human lung tissue, by fibered confocal fluorescence microscopy for the rapid identification of small molecule chemical probe-candidates with translatable potential.

Improving the speed and accuracy of bacterial detection is important for patient stratification and to ensure the appropriate use of antimicrobials. To ...

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.

Respiratory diseases in their broad diversity need appropriate model systems to understand the underlying mechanisms and enable development of new ...

Isolation and Characterization of Extracellular Vesicles from Adult Schistosoma japonicum

Extracellular vesicles (EVs) are membranous vesicles released by a variety of cells into the extracellular microenvironment. EVs represent a population of heterogeneous vesicles, whose size range between 40 and 1,000 nm. Accumulated evidence indicated that EVs play important regulatory roles in pathogen-host interactions. A deep understanding of schistosome EVs should provide insights into the mechanisms underlying schistosome-host interactions, enabling development of novel strategies against schistosomiasis. Here, we aim to further study EVs functions in schistosomes by presenting a protocol for the isolation and characterization of EVs from adult Schistosoma japonicum (S. japonicum). EVs were isolated from in vitro culture medium using centrifugation combined with a commercial exosome isolation kit. The isolated S. japonicum EVs (SjEVs) typically possess a diameter of 100 - 400 nm, and are characterized by transmission electronic microscopy and western blotting. The usage of PKH67 dye-labeled SjEVs has demonstrated that SjEVs are internalized by the recipient cells. Overall, our protocol provides an alternative method for isolating EVs from adult schistosomes; the isolated SjEVs may be suitable for functional analysis.

Isolation and Characterization of Extracellular Vesicles from Adult Schistosoma japonicum

Here, we present a protocol to describe extracellular vesicles (EVs) isolation in in vitro cultured medium from adult Schistosoma japonicum.

Extracellular vesicles (EVs) are membranous vesicles released by a variety of cells into the extracellular microenvironment. EVs represent a population of ...