Name: a tnbs-induced rodent model to study the pathogenic role of mechanical stress in crohn's disease
Uploaded: 2022-03-01T14:30:00
Description: Watch this Scientific Journal Video about A TNBS-Induced Rodent Model to Study the Pathogenic Role of Mechanical Stress in Crohn's Disease at JoVE.com

This work is supported in part by grants from NIH (R01 DK124611 to XZS) and the US Department of Defense (W81XWH-20-1-0681 to XZS). The&#160;histology work was done with the help of the UTMB Surgical Pathology Lab.

All animal experiments were conducted according to the institutional animal care and use committee of the University of Texas Medical Branch (#0907051C). Male or female Sprague-Dawley rats, ~8-9 weeks old, were used for the study.
1. Animal preparation
<ol>
	<li>Fast rats for 24 h and treat them with laxative (bowel cleanser, see Table of Materials) overnight.</li>
	<li>The next day, anesthetize rats using an anesthesia system (see Table of Materials...

<ol>
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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+mechanical+forces+on+intestinal+physiology+and+pathology.">The effects of mechanical forces on intestinal physiology and pathology.</a> Cell Signalling. 21 (8), 1237-1244 (2009).</li><li>Cox, C. S., 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=Hypertonic+saline+modulation+of+intestinal+tissue+stress+and+fluid+balance.">Hypertonic saline modulation of intestinal tissue stress and fluid balance.</a> Shock. 29 (5), 598-602 (2008).</li><li>Shi, X. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+stretch+modulates+TGF-beta1+and+alpha1(I)+collagen+expression+in+fetal+human+intestinal+smooth+muscle+cells.">Mechanical stretch modulates TGF-beta1 and alpha1(I) collagen expression in fetal human intestinal smooth muscle cells.</a> American Journal of Physiology. 277 (5), 1074-1080 (1999).</li><li>Lipson, K. E., Wong, C., Teng, Y., Spong, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CTGF+is+a+central+mediator+of+tissue+remodeling+and+fibrosis+and+its+inhibition+can+reverse+the+process+of+fibrosis.">CTGF is a central mediator of tissue remodeling and fibrosis and its inhibition can reverse the process of fibrosis.</a> Fibrogenesis Tissue Repair. 5, 24 (2012).</li><li>Chaqour, B., Goppelt-Struebe, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+regulation+of+the+Cyr61/CCN1+and+CTGF/CCN2+proteins.">Mechanical regulation of the Cyr61/CCN1 and CTGF/CCN2 proteins.</a> The FEBS Journal. 273 (16), 3639-3649 (2006).</li><li>Shi, X. Z., Winston, J. H., Sarna, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+immune+and+genetic+responses+in+rat+models+of+Crohn's+colitis+and+ulcerative+colitis.">Differential immune and genetic responses in rat models of Crohn's colitis and ulcerative colitis.</a> American Journal of Physiology-Gastrointestinal and Liver. 300 (1), 41-51 (2011).</li><li>Antoniou, 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=The+TNBS-induced+colitis+animal+model:+An+overview.">The TNBS-induced colitis animal model: An overview.</a> Annals of Medicine and Surgery (London). 11, 9-15 (2016).</li><li>Shi, X. Z., Sarna, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+therapy+of+Cav1.2+channel+with+VIP+and+VIP+receptor+agonists+and+antagonists:+A+novel+approach+to+designing+promotility+and+antimotility+agents.">Gene therapy of Cav1.2 channel with VIP and VIP receptor agonists and antagonists: A novel approach to designing promotility and antimotility agents.</a> American Journal of Physiology-Gastrointestinal and Liver. 295 (1), 187-196 (2008).</li><li>Lin, Y. M., Sarna, S. K., Shi, X. 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TNBS-induced colitis was introduced in 1989&#160;and has been used as an experimental model of Crohn&#39;s disease since then19,20,23. Significant features of this model in rodents include the development of a transmural inflammation that closely resembles the histopathological lesions developed in human Crohn&#39;s disease19,20. Previous studies on the model have focuse...

An erratum was issued for:&#160;<a href="https://www.jove.com/t/63499">A TNBS-Induced Rodent Model to Study the Pathogenic Role of Mechanical Stress in Crohn&#39;s Disease</a>. The Introduction and Protocol were updated.
The first sentence of the Introduction was updated from:
Inflammatory bowel diseases (IBD) such as Crohn&#39;s disease (CD) are chronic inflammatory disorders of the gastrointestinal tract affecting approximately 20 per 1,00,000 in Europe and USA. CD is characterized by transmural inflammation, intestinal fibrosis, and luminal stenosis.
to:
Inflammatory bowel diseases (IBD) such as Crohn&#39;s disease (CD) are chronic inflammatory disorders of the gastrointestinal tract affecting approximately 20 per 1,000,000 in Europe and USA. CD is characterized by transmural inflammation, intestinal fibrosis, and luminal stenosis.
Step 1.3 in the Protocol was updated from:
Prepare fresh TNBS solution according to body weights. 
NOTE: TNBS - 65 mg/kg of body weight in 250 &#181;L of 40% ethanol was used.
to:
Prepare fresh TNBS solution according to body weights. 
NOTE: TNBS - 65 mg/kg of body weight in 250 &#181;L of 40% ethanol/saline was used.

An erratum was issued for:&#160;<a href="https://www.jove.com/t/63499">A TNBS-Induced Rodent Model to Study the Pathogenic Role of Mechanical Stress in Crohn&#39;s Disease</a>. The Introduction and Protocol were updated.

Erratum: A TNBS-Induced Rodent Model to Study the Pathogenic Role of Mechanical Stress in Crohn's Disease

Inflammatory bowel disease (IBD), including ulcerative colitis (UC) and Crohn&#39;s disease (CD), is characterized by chronic inflammation in the gastrointestinal (GI) tract. It affects ~1-2 million Americans1. The estimated annual costs for IBD care in the US are $11.8 billion. Unlike UC, the CD is characterized by transmural inflammation and stricture formation2,3. Stricture formation (stenosis) occurs in up to 70% of CD patients3 and may be caused by transmural inflammation (inflammatory stenosis) or intestinal fibrosis (fibrotic stenosis)4,5. Intestinal fibrosis is characterized by excessive collagen deposition and other extracellular matrices (ECM) with smooth muscle cells (SMC) as one of the main mesenchymal cell types involved in the process3,4. Smooth muscle hyperplasia associated with hypertrophy is another significant histological change in fibrotic stenosis in CD6. Although stricture formation in CD is associated with chronic inflammation, no anti-inflammatory treatment is effective, except surgical treatment2,6. However, post-surgery recurrences are almost 100%, given sufficient time2,7. As an inflammatory response, fibrosis and SMC hyperplasia may also develop in non-inflammatory conditions (i.e., bowel obstruction) in the gut8,9; it is believed that both inflammation-dependent and independent mechanisms are involved in stricture formation3,4. Given that extensive research into the inflammation-dependent mechanisms has not translated into any effective therapy for stricture formation, studies into the possible role of inflammation-independent mechanisms in intestinal fibrosis are needed.
As a non-inflammatory factor, mechanical stress (MS) associated with edema, inflammatory cell infiltration, tissue deformation, fibrosis, and stenosis10,11,12,13 is commonly encountered in IBD, especially CD, which is characterized by transmural inflammation. Mechanical stress is most remarkable in stenotic CD, where stenosis (inflammatory or fibrotic) in the inflammation site presents mechanical stress in the local tissue and leads to lumen distention in the segment proximal to the obstruction site10,14. Previous in vitro studies have demonstrated that mechanical stress alters gene expression of specific inflammatory mediators (i.e., COX-2, IL-6)8,14,15 and growth factors (i.e., TGF-&#946;) in the gastrointestinal tissues, especially gut smooth muscle cells (SMC)16. Recent studies also found that the expression of specific pro-fibrotic mediators such as connective tissue growth factor (CTGF) is highly sensitive to mechanical stress17,18. It was hypothesized that mechanical stress might play an independent pathogenic role in CD-associated inflammation, fibrosis, and tissue remodeling. However, the pathogenic significance of mechanical stress in gut inflammation, fibrosis, and smooth muscle hyperplasia in CD remains largely unexplored. This may be partly because inflammation is a more visible and better-studied process than mechanical stress. More importantly, there has been no well-defined animal model of IBD to distinguish the effect of mechanical stress from that of inflammation.
The current work describes a rodent model of Crohn&#39;s-like colitis induced by intracolonic injection of hapten reagent 2,4,6-trinitrobenzene sulfonic acid (TNBS)19,20, which may serve the purpose to study the role of mechanical stress in CD. It was found that TNBS instillation induced a localized (~2 cm in length) transmural inflammation with lumen narrowing (stenosis) in the distal colon. The stenosis leads to marked bowel distention (mechanical stress)14,15 but not visible inflammation in the colonic segment proximal to the instillation site. On the contrary, the colon segment distal to the stenosis site presents neither inflammation nor mechanical stress. Significant site-specific changes in gene expression, inflammation, fibrosis, and SMC hyperplasia were observed in the three different sites. The results suggest that mechanical stress, particularly mechanical stress-induced gene expression, may play a critical role in developing fibrosis and hyperplasia in Crohn&#39;s colitis.

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			<td>ACT-1 Control Software Ver2.63</td>
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			<td>C1000 Touch Thermal Cycler with 96-Well Fast Reaction Module</td>
			<td>BIO-RAD</td>
			<td>1851196</td>
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			<td>CFX96 Optical Reaction Module for Real-Time PCR Systems</td>
			<td>BIO-RAD</td>
			<td>1845097</td>
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			<td>Dako Agilent Artisan Link Pro Special stainer</td>
			<td>Dako</td>
			<td>AR310</td>
			<td></td>
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			<td>Dako-Agilent Masson&#39;s Trichrome Kit ref# AR173</td>
			<td>Dako</td>
			<td>AR173</td>
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			<td>DXM1200 Digital Color HR Camera</td>
			<td>Nikon</td>
			<td>DXM1200</td>
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			<td>Eukaryotic 18S rRNA Endogenous Control</td>
			<td>ThermoFisher Scientific</td>
			<td>4352930E</td>
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			<td>E-Z Anesthesia</td>
			<td>E-Z Systems Inc.</td>
			<td>EZ-155</td>
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			<td>GraphPad Prism 9</td>
			<td>GraphPad</td>
			<td>9.0.2 (161)</td>
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			<td>Hard-Shell 96-Well PCR Plates, low profile, thin wall, skirted, white/clear</td>
			<td>BIO-RAD</td>
			<td>HSP9601</td>
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			<td>HBSS (Corning Hank&#39;s Balanced Salt Solution, 1x without calcium and magnesium)</td>
			<td>CORNING</td>
			<td>21-021-CV</td>
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			<td>HM 325 Microtome</td>
			<td>Thermo Scientific</td>
			<td>23-900-667</td>
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			<td>Isoflurane</td>
			<td>Piramal</td>
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			<td>LI-COR Odyssey Digital Imaging System</td>
			<td>LI-COR</td>
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			<td>Mastercycler epGradient Thermal Cycler with Control Panel 5340 Thermal Cycler</td>
			<td>Eppendorf</td>
			<td>5341</td>
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			<td>Medical grade open end polyurethane catheter</td>
			<td>Covidien</td>
			<td>8890703013</td>
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			<td>NanoDrop 2000/2000c Spectrophotometers</td>
			<td>Thermo Fisher Scientific</td>
			<td>ND2000CLAPTOP</td>
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			<td>Nikon Eclipse E800 Upright Microscope</td>
			<td>Nikon</td>
			<td>E800</td>
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			<td>Nitrocellulose/Filter Paper Sandwiches Pkg of 50, 0.45 &#956;m, 7 x 8.5 cm</td>
			<td>BIO-RAD</td>
			<td>1620215</td>
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			<td>Polyethylene Glycol 3350, Osmotic Laxative</td>
			<td>Miralax</td>
			<td>C8175</td>
			<td>Dose: 17g in 226 mL of water</td>
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			<td>RNeasy Mini Kit (250) 
			250 RNeasy Mini Spin Columns, Collection Tubes (1.5 mL and 2 mL), RNase-free Reagents and Buffers</td>
			<td>QIAGEN</td>
			<td>74106</td>
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			<td>SuperScript III First-Strand Synthesis System</td>
			<td>ThermoFisher Scientific</td>
			<td>18080051</td>
			<td></td>
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			<td>TaqMan Gene Expression Assays Rn00573960_g1 CTGF Probe</td>
			<td>ThermoFisher Scientific</td>
			<td>4331182</td>
			<td></td>
		</tr>
		<tr>
			<td>TaqMan Gene Expression Assays Rn99999011_m1 IL6 Probe</td>
			<td>ThermoFisher Scientific</td>
			<td>4331182</td>
			<td></td>
		</tr>
		<tr>
			<td>TaqMan Fast Advanced Master Mix</td>
			<td>ThermoFisher Scientific</td>
			<td>4444557</td>
			<td></td>
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			<td>Tissue-Tek Prisma H&#38;E Stain Kit #1</td>
			<td>Sakura</td>
			<td>6190</td>
			<td></td>
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			<td>Tissue-Tek Prisma Plus Automated Slide Stainer</td>
			<td>Sakura</td>
			<td>6171</td>
			<td></td>
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			<td>TNBS (Picrylsulfonic acid solution)</td>
			<td>SIGMA-ALDRICH</td>
			<td>92822</td>
			<td></td>
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a tnbs-induced rodent model to study the pathogenic role of mechanical stress in crohn's disease

Inflammatory bowel diseases (IBD) such as Crohn&#39;s disease (CD) are chronic inflammatory disorders of the gastrointestinal tract affecting approximately 20 per 1,000,000 in Europe and USA. CD is characterized by transmural inflammation, intestinal fibrosis, and luminal stenosis. Although anti-inflammatory therapies may help control inflammation, they have no efficacy on fibrosis and stenosis in CD. The pathogenesis of CD is not well understood. Current studies focus mainly on delineating dysregulated gut immune response mechanisms. While CD-associated transmural inflammation, intestinal fibrosis, and luminal stenosis all represent mechanical stress to the gut wall, the role of mechanical stress in CD is not well defined. To determine if mechanical stress plays an independent pathogenic role in CD, a protocol of TNBS-induced CD-like colitis model in rodents has been developed. This TNBS-induced transmural inflammation and fibrosis model resembles pathological hallmarks of CD in the colon. It is induced by intracolonic instillation of TNBS into the distal colon of adult Sprague-Dawley rats. In this model, transmural inflammation leads to stenosis at the TNBS instillation site (Site I). Mechanical distention is observed in the portion proximal to the instillation site (Site P), representing mechanical stress but not visible inflammation. Colonic portion distal to inflammation (Site D) presents neither inflammation nor mechanical stress. Distinctive changes of gene expression, immune response, fibrosis, and smooth muscle growth at different sites (P, I, and D) were observed, highlighting a profound impact of mechanical stress. Therefore, this model of CD-like colitis will help us better understand CD&#39;s pathogenic mechanisms, particularly the role of mechanical stress and mechanical stress-induced gene expression in immune dysregulation, intestinal fibrosis, and tissue remodeling in CD.

Macroscopic view of Crohn&#39;s-like colitis induced by intra-colonic instillation of TNBS 
As shown in Figure 1, intracolonic instillation of TNBS in rats induced a localized transmural inflammation (~2 cm in length) with thickened bowel wall and narrowed lumen (stenosis) in the site of instillation in the distal colon (Figure 1A). The site of TNBS instillation is referred to as site I. As a result of transmural inflammation and stenosis, ...

Watch this Scientific Journal Video about A TNBS-Induced Rodent Model to Study the Pathogenic Role of Mechanical Stress in Crohn's Disease at JoVE.com

A TNBS-Induced Rodent Model to Study the Pathogenic Role of Mechanical Stress in Crohn&#039;s Disease

The present protocol describes the development of a Crohn's-like colitis model in rodents. Transmural inflammation leads to stenosis at the TNBS instillation site, and mechanical enlargement is observed in the segment proximal to the stenosis. These changes allow studying mechanical stress in colitis.

A TNBS-Induced Rodent Model to Study the Pathogenic Role of Mechanical Stress in Crohn's Disease

Inflammatory bowel diseases (IBD) such as Crohn&#39;s disease (CD) are chronic inflammatory disorders of the gastrointestinal tract affecting ...

So in our protocol, intra-colonic instillation of TNBS leads to transmural inflammation at the instillation site and the mechanical distention in the segment proximal to inflammation. So this mimics stenotic Crohn's disease. As the proximal segment is mechanically distended but does not show a visible inflammation, a comparative study of the proximal segment and the inflammation site helps to determine if mechanical stress plays a role in Crohn's colitis.Previous studies focus on the inflammation site only. Our protocol working on different sites in the model highlights a critical role of mechanical stress in Crohn's disease, particularly in stenotic Crohn's disease. To begin, place the rats on the anesthesia table in a supine position.To induce colitis, insert a medical-grade, open-end, polyurethane catheter through the anus for approximately seven to eight centimeters from the anal verge, and gently instill TNBS into the colon. Administer the sham control rats with 250 microliters of saline. To help the distribution of TNBS or saline and avoid spills, keep the rats in a supine position with their head facing downwards at approximately a 30-degree angle, with the anus closed for two minutes.Open the rat abdomen using surgical-grade scissors and forceps. Carefully remove the entire colon above the anal canal, and transfer the colon immediately to ice-cold 1x HBSS buffer. Straighten the colon in the buffer, and measure the colon length using a ruler.Take a nylon thread, and circle it around the colon to measure the external circumference of the colon segments in control and TNBS-treated rats. Take full-thickness tissues for histology. Cut open the colon along the mesenteric board, and clean the colon thoroughly with HBSS buffer.Assess the colon for macroscopic inflammation score based on the predefined criteria with minimal modifications. Collect colonic tissue samples from TNBS-treated rats at sites P, I, and D.Site P is the portion two to three centimeters before the oral margin of the inflammation site. Site I is the inflammation site where TNBS is instilled, typically four to six centimeters from the end of the colon.Finally, site D is the portion one to two centimeters distal to the aboral margin of the inflammation site. Intra-colonic instillation of TNBS in rats induced a localized transmural inflammation approximately two centimeters in length with thickened bowel wall and narrowed lumen in the site of instillation in the distal colon. The site of TNBS instillation was referred to as site I.As a result of transmural inflammation and stenosis, both inflammation and mechanical stress were present in site I.The stenosis in site I led to marked lumen distension in the segment proximal to the site of TNBS instillation, referred to as site P.The colon circumference was significantly increased in sites P and I compared to sham control colon.While mechanically distended, site P did not show visible inflammation. On the contrary, the colonic segment distal to the TNBS instillation referred to as site D presented neither inflammation nor mechanical distension. The macroscopic score of inflammation in site I in the TNBS-treated rats was dramatically increased compared to sham controls in sites P and D of colitis rats.The inflammation scores in sites P and D were not significantly increased compared to sham. So it is important to study not just site I but sites P and D in the model, as this helps to recognize the pathogenic significance of mechanical stress in Crohn's disease. To further determine if mechanical stress is a pathogenic factor in gut inflammation, one may also try to release mechanical distention by feeding the animals exclusively with liquid diet and see if this helps with inflammation and bowel function.

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Research

JoVE Journal

Immunology and Infection

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 ...

Cutaneous Leishmaniasis in the Dorsal Skin of Hamsters: a Useful Model for the Screening of Antileishmanial Drugs

Traditionally, hamsters are experimentally inoculated in the snout or the footpad. However in these sites an ulcer not always occurs, measurement of lesion size is a hard procedure and animals show difficulty to eat, breathe and move because of the lesion. In order to optimize the hamster model for cutaneous leishmaniasis, young adult male and female golden hamsters (Mesocricetus auratus) were injected intradermally at the dorsal skin with 1 to 1.5 x l07 promastigotes of Leishmania species and progression of subsequent lesions were evaluated for up to 16 weeks post infection. The golden hamster was selected because it is considered the adequate bio-model to evaluate drugs against Leishmania as they are susceptible to infection by different species. Cutaneous infection of hamsters results in chronic but controlled lesions, and a clinical evolution with signs similar to those observed in humans. Therefore, the establishment of the extent of infection by measuring the size of the lesion according to the area of indurations and ulcers is feasible. This approach has proven its versatility and easy management during inoculation, follow up and characterization of typical lesions (ulcers), application of treatments through different ways and obtaining of clinical samples after different treatments. By using this method the quality of animal life regarding locomotion, search for food and water, play and social activities is also preserved.

Optimization of the experimental hamster model for cutaneous leishmaniasis by intradermal injection of Leishmania promastigotes at the dorsal skin. This approach is useful during inoculation, follow-up, characterization of lesions, application of treatments and obtaining of clinical samples. Locomotion, search for food and water, play and social activities are preserved.

Traditionally, hamsters are experimentally inoculated in the snout or the footpad. However in these sites an ulcer not always occurs, measurement of ...

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 ...

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 ...

Examining the Role of Nasopharyngeal-associated Lymphoreticular Tissue (NALT) in Mouse Responses to Vaccines

The nasopharyngeal-associated lymphoreticular tissues (NALT) found in humans, rodents, and other mammals, contribute to immunity in the nasal sinuses1-3. The NALT are two parallel bell-shaped structures located in the nasal passages above the hard palate, and are usually considered to be secondary components of the mucosal-associated lymphoid system4-6. Located within the NALT are discrete compartments of B and T lymphocytes interspersed with antigen-presenting dendritic cells4,7,8. These cells are surrounded by an epithelial cell layer intercalated with M-cells that are responsible for antigen retrieval from the mucosal surfaces of the air passages9,10. Naive lymphocytes circulating through the NALT are poised to respond to first encounters with respiratory pathogens7. While NALT disappear in humans by the age of two years, the Waldeyer's Ring and similarly structured lymphatic organs continue to persist throughout life6. In contrast to humans, mice retain NALT throughout life, thus providing a convenient animal model for the study of immune responses originating within the nasal sinuses11.
Cultures of single-cell suspensions of NALT are not practical due to low yields of mononuclear cells. However, NALT biology can be examined by ex vivo culturing of the intact organ, and this method has the additional advantage of maintaining the natural tissue structure. For in vivo studies, genetic knockout models presenting defects limited to NALT are not currently available due to a poor understanding of the developmental pathway. For example, while lymphotoxin-&alpha; knockout mice have atrophied NALT, the Peyer's patches, peripheral lymph nodes, follicular dendritic cells and other lymphoid tissues are also altered in these genetically manipulated mice12,13. As an alternative to gene knockout mice, surgical ablation permanently eliminates NALT from the nasal passage without affecting other tissues. The resulting mouse model has been used to establish relationships between NALT and immune responses to vaccines1,3. Serial collection of serum, saliva, nasal washes and vaginal secretions is necessary for establishing the basis of host responses to vaccination, while immune responses originating directly from NALT can be confirmed by tissue culture. The following procedures outline the surgeries, tissue culture and sample collection necessary to examine local and systemic humoral immune responses to intranasal (IN) vaccination.

Examining the Role of Nasopharyngeal-associated Lymphoreticular Tissue NALT in Mouse Responses to Vaccines

Methods to examine contributions of the nasopharyngeal-associated lymphoreticular tissues (NALT) to nasal and systemic immune responses of mice to intranasal vaccines are described. We demonstrate a surgical procedure to establish a NALT-dependent mouse model and ex vivo cultures of extracted NALT.

The nasopharyngeal-associated lymphoreticular tissues (NALT) found in humans, rodents, and other mammals, contribute to immunity in the nasal sinuses1-3. ...

An In vitro Co-infection Model to Study Plasmodium falciparum-HIV-1 Interactions in Human Primary Monocyte-derived Immune Cells

Plasmodium falciparum, the causative agent of the deadliest form of malaria, and human immunodeficiency virus type-1 (HIV-1) are among the most important health problems worldwide, being responsible for a total of 4 million deaths annually1. Due to their extensive overlap in developing regions, especially Sub-Saharan Africa, co-infections with malaria and HIV-1 are common, but the interplay between the two diseases is poorly understood. Epidemiological reports have suggested that malarial infection transiently enhances HIV-1 replication and increases HIV-1 viral load in co-infected individuals2,3. Because this viremia stays high for several weeks after treatment with antimalarials, this phenomenon could have an impact on disease progression and transmission.
The cellular immunological mechanisms behind these observations have been studied only scarcely. The few in vitro studies investigating the impact of malaria on HIV-1 have demonstrated that exposure to soluble malarial antigens can increase HIV-1 infection and reactivation in immune cells. However, these studies used whole cell extracts of P. falciparum schizont stage parasites and peripheral blood mononuclear cells (PBMC), making it hard to decipher which malarial component(s) was responsible for the observed effects and what the target host cells were4,5. Recent work has demonstrated that exposure of immature monocyte-derived dendritic cells to the malarial pigment hemozoin increased their ability to transfer HIV-1 to CD4+ T cells6,7, but that it decreased HIV-1 infection of macrophages8. To shed light on this complex process, a systematic analysis of the interactions between the malaria parasite and HIV-1 in different relevant human primary cell populations is critically needed.
Several techniques for investigating the impact of HIV-1 on the phagocytosis of micro-organisms and the effect of such pathogens on HIV-1 replication have been described. We here present a method to investigate the effects of P. falciparum-infected erythrocytes on the replication of HIV-1 in human primary monocyte-derived macrophages. The impact of parasite exposure on HIV-1 transcriptional/translational events is monitored by using single cycle pseudotyped viruses in which a luciferase reporter gene has replaced the Env gene while the effect on the quantity of virus released by the infected macrophages is determined by measuring the HIV-1 capsid protein p24 by ELISA in cell supernatants.

An In vitro Co-infection Model to Study Plasmodium falciparum-HIV-1 Interactions in Human Primary Monocyte-derived Immune Cells

We have developed an in vitro malaria-HIV-1 co-infection model to study the impact of Plasmodium falciparum on the HIV-1 replicative cycle in human primary monocyte-derived macrophages. This versatile system can easily be adapted to other primary cell types susceptible to HIV-1 infection.

Plasmodium falciparum, the causative agent  of the deadliest form of malaria, and human immunodeficiency virus type-1 (HIV-1) are among the most important ...

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the phenotypic and functional differences between murine and human monocyte-derived macrophages. Therefore, we here describe an in vitro model to generate and study primary human macrophages. Briefly, after density gradient centrifugation of peripheral blood drawn from a forearm vein, monocytes are isolated from peripheral blood mononuclear cells using negative magnetic bead isolation. These monocytes are then cultured for six days under specific conditions to induce different types of macrophage differentiation or polarization. The model is easy to use and circumvents the problems caused by species-specific differences between mouse and man. Furthermore, it is closer to the in vivo conditions than the use of immortalized cell lines. In conclusion, the model described here is suitable to study macrophage biology, identify disease mechanisms and novel therapeutic targets. Even though not fully replacing experiments with animals or human tissues obtained post mortem, the model described here allows identification and validation of disease mechanisms and therapeutic targets that may be highly relevant to various human diseases.

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Monocyte-derived macrophages are important cells of the innate immune system. Here, we describe an easy to use in vitro model to generate these cells. Using gradient centrifugation, negative bead isolation and specific cell culture conditions, monocyte-derived macrophages can be generated for phenotypic and functional studies.

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the ...

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 ...

An Ex Vivo Chicken Primary Bursal-cell Culture Model to Study Infectious Bursal Disease Virus Pathogenesis

Infectious bursal disease virus (IBDV) is a birnavirus of economic importance to the poultry industry. The virus infects B cells, causing morbidity, mortality, and immunosuppression in infected birds. In this study, we describe the isolation of chicken primary bursal cells from the bursa of Fabricius, the culture and infection of the cells with IBDV, and the quantification of viral replication. The addition of chicken CD40 ligand significantly increased cell proliferation fourfold over six days of culture and significantly enhanced cell viability. Two strains of IBDV, a cell-culture adapted strain, D78, and a very virulent strain, UK661, replicated well in the ex vivo cell cultures. This model will be of use in determining how cells respond to IBDV infection and will permit a reduction in the number of infected birds used in IBDV pathogenesis studies. The model can also be expanded to include other viruses and could be applied to different species of birds.

An Ex Vivo Chicken Primary Bursal-cell Culture Model to Study Infectious Bursal Disease Virus Pathogenesis

Here we describe the isolation of chicken primary bursal cells from the bursa of Fabricius, the culture and infection of the cells with infectious bursal disease virus, and the quantification of viral replication.

Infectious bursal disease virus (IBDV) is a birnavirus of economic importance to the poultry industry. The virus infects B cells, causing morbidity, ...

Production of Pseudotyped Particles to Study Highly Pathogenic Coronaviruses in a Biosafety Level 2 Setting

The protocol aims to generate coronavirus (CoV) spike (S) fusion protein pseudotyped particles with a murine leukemia virus (MLV) core and luciferase reporter, using a simple transfection procedure of the widely available HEK-293T cell line. Once formed and released from producer cells, these pseudovirions incorporate a luciferase reporter gene. Since they only contain the heterologous coronavirus spike protein on their surface, the particles behave like their native coronavirus counterparts for entry steps. As such, they are the excellent surrogates of native virions for studying viral entry into host cells. Upon successful entry and infection into target cells, the luciferase reporter gets integrated into the host cell genome and is expressed. Using a simple luciferase assay, transduced cells can be easily quantified. An important advantage of the procedure is that it can be performed in biosafety level 2 (BSL-2) facilities instead of BSL-3 facilities required for work with highly pathogenic coronaviruses such as Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus (SARS-CoV). Another benefit comes from its versatility as it can be applied to envelope proteins belonging to all three classes of viral fusion proteins, such as the class I influenza hemagglutinin (HA) and Ebola virus glycoprotein (GP), the class II Semliki forest virus E1 protein, or the class III vesicular stomatitis virus G glycoprotein. A limitation of the methodology is that it can only recapitulate virus entry steps mediated by the envelope protein being investigated. For studying other viral life cycle steps, other methods are required. Examples of the many applications these pseudotype particles can be used in include investigation of host cell susceptibility and tropism and testing the effects of virus entry inhibitors to dissect viral entry pathways used.

Here, we present a protocol to generate pseudotyped particles in a BSL-2 setting incorporating the spike protein of the highly pathogenic viruses Middle East respiratory syndrome and severe acute respiratory syndrome coronaviruses. These pseudotyped particles contain a luciferase reporter gene allowing quantification of virus entry into target host cells.

The protocol aims to generate coronavirus (CoV) spike (S) fusion protein pseudotyped particles with a murine leukemia virus (MLV) core and luciferase ...