The research leading to these results has received funding from the People Program (Marie Curie Actions) under REA grant agreement number 302170 of the European Union&#180;s Seventh Framework Programme (FP7/2007-2013); the Interdisciplinary Center for Clinical Research (IZKF) of the University Erlangen-Nuremberg; the Collaborative Research Center TRR241 and the Clinical Research Group KFO257 of the German Research Council (DFG); and the DFG.

The following protocol has been approved by the relevant local authorities in Erlangen (Regierung von Unterfranken, W&#252;rzburg, Germany). Mice were housed under specific pathogen-free conditions.
NOTE: Inhibition of GGTase-mediated prenylation within IECs causes a severe alteration of intestinal permeability in Pggt1bi&#916;IEC mice24. Therefore, this mouse model was used to demonstrate how the protocol can be useful to study intestinal barrier defects. However, this protocol could be used for the study of any other mouse line.
1. Surgical preparation and mouse intestinal mucosa staining
NOTE: The surgical preparation is based on previously described protocols25. Keep the anesthetized mouse under a red lamp during the surgical preparation, to avoid a drop in body temperature.
<ol>
	<li>Anesthetize mouse by intraperitoneal injection of ketamine/xylazin (96 mg/kg ketamine; and 12.8 mg/kg xylazin). Verify the anesthesia by checking for the lack of an eyelid reflex.</li>
	<li>Apply eye protection cream using a cotton bud.</li>
	<li>Make an incision (1 cm) on the left ventral area using standard forceps and straight fine scissors.</li>
	<li>Exteriorize a segment of the intestine (5-7 cm, approximately).</li>
	<li>Open the exteriorized intestinal segment longitudinally by electrocauterization at the antimesenteric side.</li>
	<li>Expose the mucosa and rinse shortly with saline solution to remove fecal content. 
	NOTE: Optionally, apply xylazin directly on the gut to avoid motion artifacts due to peristalsis.</li>
	<li>Stain the surface of the intestinal mucosa with acriflavine and rhodamine-B dextran (10 kDa).
	<ol>
		<li>Apply the 1 mg/mL acriflavine solution (100 &#181;L) by pipetting drop by drop on the mucosa and incubate for 3 min. Wash out the remaining solution with PBS.</li>
		<li>Apply the 2 mg/mL rhodamine-dextran solution (100 &#181;L) by pipetting drop by drop on the mucosa and incubate for 3 min. Wash out the remaining solution with PBS. 
		NOTE: Optionally, remove blood from the preparation using aseptic cotton.</li>
	</ol>
	</li>
	<li>Place the anesthetized mouse supine on a cover slide mounted in a chamber rinsed with pre-warmed saline solution (37 &#176;C). 
	NOTE: The opened intestinal segment is then placed luminal surface down, on the cover slide.</li>
	<li>Place the preparation (anesthetized mouse in the chamber) on the inverted microscope stage.</li>
	<li>Cover the animal with an isothermal pad (approx. 37 &#176;C).</li>
	<li>Proceed immediately to intravital microscopy. 
	NOTE: Keep the surgical preparation rinsed with pre-warmed saline solution to avoid dehydration of the tissue and cell death.</li>
</ol>
2. Intravital microscopy
NOTE: Perform step 2.1 before starting the surgical preparation, to avoid long waiting times between anesthesia, surgery and image acquisition. If necessary, additional doses of anesthetics can be given to surgically prepared animals to keep them under anesthesia for the imaging experiments.
<ol>
	<li>Set up the CLSM microscope.
	<ol>
		<li>Start the CLSM microscope by turning on the microscope base and the scanner box. Turn on the computer by pressing the Start button.</li>
		<li>Launch the image acquisition software (e.g., LAS X) by double clicking on the icon. Select the appropriate configuration (Configuration: machine; Microscope: DMI6000) and click OK.
		<ol>
			<li>Define the appropriate Resolution. Go to Configuration | Hardware | Resolution | Bit depth. Select 12.</li>
		</ol>
		</li>
		<li>Go to the Acquisition menu. Select xyzt for the image acquisition mode from the drop-down menu. Select the objective from the drop-off menu (20x or 40x).</li>
		<li>Design the sequential acquisition setting. Click on Seq. Add a second sequence, by clicking on the add button (+). Select Between frames.
		<ol>
			<li>Configure Sequence 1 (detection of acriflavine; 416 nm excitation; 514 nm emission). Turn on the visible laser box. Activate the PMT1 (ON). Define the emission wavelength window (490-550 nm).</li>
			<li>Configure Sequence 2 (detection of rhodamineB-dextran; 570 nm excitation; 590 nm, emission). Turn on the visible laser box. Activate the PMT2 (ON). Define the emission wavelength window (550-760).</li>
		</ol>
		</li>
		<li>Activate the corresponding lasers (488 and 552). Go to Configuration | Lasers. Activate 488 and 552 nm lasers (turn ON).</li>
	</ol>
	</li>
	<li>Adjust the setting to the specific features of the current experiment.
	<ol>
		<li>Turn ON the light source (press power). Place the preparation (anesthetized mouse in the chamber) on the microscope stage and change the xy position until the illumination axis is focused on the tissue preparation.</li>
		<li>Select the field of interest using the standard light source and the eyepieces.
		<ol>
			<li>Select the filter cube (I3). Open the shutter. Focus on the surface of the intestinal mucosa by using the macro- and micro-wheel. Search for an area where several villi can be visualized within the field of view by changing the XY position.</li>
		</ol>
		</li>
		<li>Verify that the rhodamine-dextran staining is also visible in that area. Change the filter cube (N2.1). Check the image through the eyepieces.</li>
		<li>Start the CLSM image acquisition from the software. Optimize settings for the two sequences.
		<ol>
			<li>Select Sequence 1. Adjust laser power, gain and offset for Sequence 1.</li>
			<li>Select Sequence 2. Adjust laser power, gain and offset for Sequence 2.</li>
		</ol>
		</li>
		<li>Define the z stack range.
		<ol>
			<li>Open the Z-stack drop-off menu. Focus on the surface of the mucosa using the z-axis control. Press Begin.</li>
			<li>Focus on the bottom limit where the signal is still detectable. Press End.</li>
			<li>Define the numbers of z stacks (10). Avoid time lapses longer than 2 min for two consecutive time points.</li>
		</ol>
		</li>
		<li>Define the time settings. Go to the Time menu. Click Minimize. Select Acquire until stopped.</li>
		<li>Define the line average. Select Seq 1. Select Line Average 2. Select Seq 2. Select Line Average 2.</li>
	</ol>
	</li>
	<li>Acquire corresponding images.
	<ol>
		<li>Select Format (1024 x 1024) and Speed (400). Press Start.</li>
		<li>Press Stop after the desired image acquisition time.</li>
	</ol>
	</li>
	<li>Save the file. Go to Project. Right click on the corresponding file. Press Save as.
	<ol>
		<li>Name the file appropriately. Select the adequate folder. Press Save.</li>
	</ol>
	</li>
	<li>Euthanize the animal (cervical dislocation) and collect tissues for ulterior analysis, if needed.</li>
</ol>
3. Image analysis: Determine cell shedding rate and the intestinal epithelium
<ol>
	<li>Cell shedding rate
	<ol>
		<li>Launch the image acquisition software.</li>
		<li>Go to Open projects. Select the appropriate file.</li>
		<li>Define the total time of image acquisition.
		<ol>
			<li>Select the last complete z stack. Select the last Z position from the previously selected time point. Read the time on the right bottom corner of the screen (total time of image acquisition).</li>
		</ol>
		</li>
		<li>Select a villus.
		<ol>
			<li>Select the appropriate Z stack position (surface of the villus, but deep enough to avoid the interference with already shed cells and other components present at the lumen).</li>
			<li>Measure the length of basal membrane. Select the ruler tool. Divide the villus in several lines to cover or draw the whole villus perimeter. Add the different values (total length of the basal membrane). Delete the segments of the Ruler tool.</li>
		</ol>
		</li>
		<li>Count the number of events occurring during the whole duration of the image acquisition.
		<ol>
			<li>Divide the villus into segments with an adequate size. Analyze the sequence of events/segment by sliding the bar through the different time points. Annotate the identified cell shedding events.</li>
		</ol>
		</li>
		<li>Calculate the number of shedding events/time/length of basal membrane, which indicates the cell shedding rate. Count up to 5 villi/video, and at least two videos/mouse. Calculate the mean of these 10 measurements.</li>
	</ol>
	</li>
	<li>Leakage
	<ol>
		<li>Select a villus.</li>
		<li>Select the appropriate Z stack position (surface of the villus, but deep enough to avoid the interference with already shed cells and other components present at the lumen). Count total number of epithelial cells/villus.</li>
		<li>Count the number of leakage points (Para-cellular presence of rhodamine dextran. This event is transitory, which can be confirmed by checking previous and ulterior acquired pictures).</li>
		<li>Calculate the number of leakage/total number of epithelial cells. Analyze 10 different villus/sample/video and calculate the average number of leakage and permeable cells/villus.</li>
	</ol>
	</li>
</ol>

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Although technically challenging, intravital microscopy-based methodology represents a unique experimental approach to visualize highly dynamic cellular process in real time, such as cell shedding performance. Thus far, there is no alternative experimental approach to visualize cell extrusion in vivo. We believe that this protocol can contribute to the description of diverse cellular processes playing a role in the maintenance of intestinal homeostasis.
Taking advantage of intravital microscop...

The intestine is a highly specialized organ with a tightly regulated function enabling conflicting processes, namely nutrition and protection against harmful luminal substances. Lining between the human body and the environment, the intestinal epithelium acts as a physical and immunological barrier and contributes to the maintenance of mucosal homeostasis in the gut1,2. Loss of epithelial integrity and increased tight junction permeability is well known to be associated with Inflammatory Bowel Disease (IBD)3,4,5,6. Epithelial alterations are then considered as causes and secondary amplifiers for chronic intestinal inflammation in IBD. Thus, an improved understanding of early epithelial alterations in the gut of IBD patients would be of immense value for the development of new strategies to restore epithelial integrity for reliable prediction and subsequent prevention of IBD relapses.
Intestinal epithelium follows a complex and tightly regulated turnover process. From the crypt bottom, terminally differentiated intestinal epithelial cells (IECs) derived from pluripotent stem cells migrate upwards to the villus tip, where aged/damaged cells are shed into the lumen7. The equilibrium between division and cell extrusion enables the maintenance of intestinal epithelial cell numbers, avoiding the formation of gaps and leakage, as well as the accumulation of epithelial cells potentially leading to cell masses and tumorigenesis8,9,10. Despite the key role of epithelial cell shedding in the physiological renewal of the gut epithelium, the knowledge about the molecular mechanisms driving the extrusion of cells at the villus tip is limited. Thus, there is a need for basic research providing a precise description of the sequence of molecular events involved in epithelial cell shedding.
Complex interactions between different cell types within the intestinal mucosa are key to understand the molecular mechanisms regulating epithelial turnover and intestinal homeostasis. Thus, in vivo studies offer high advantages over in vitro and ex vivo approaches in this context. Moreover, real-time imaging techniques permit the description of the sequence of events controlling specific phenomena. In this context, the study of highly dynamic processes demands the use of optimized high resolution techniques for the direct observation of the tissue. Intravital imaging techniques appear as unique suitable tools for the study of epithelial cell shedding in the gut.
The term &#34;intravital microscopy&#34; refers to experimental approaches taking advantage of high-resolution imaging techniques (multiphoton or confocal microscopy) to directly visualize cells and tissues in their native environs within a living animal11. It enables real time acquisition of in vivo information up to single-cell resolution, and entails clear advantages over static or low resolution methods. Intravital microscopy provides complementary information and overcome some limitations from classical and/or high-end techniques, such as artifacts due to tissue processing. In contrast, the main limitation of intravital microscopy is that the tissue should be directly exposed to the microscope, which in most cases requires surgery. Although sophisticated approaches preserve the vitality and minimize the impact of the imaged tissue (skinfold chambers and imaging windows)12,13, in most cases a simple skin incision is performed for the externalization of the tissue (skin flaps)14. In the last decade, these approaches have contributed key evidence about highly dynamic processes, which were previously inscrutable. Translationally, real-time imaging provided new biological insights on stem cell and leukocytes homing15, as well as cancer dissemination and metastasis formation13,16. In the clinical context, endomicroscopy is currently exploited as a diagnostic tool of cancer17 and gastrointestinal diseases, such as IBD18,19; while confocal mosaicking microscopy became a rapid pathology tool during surgery20. Together, intravital microscopy has lately emerged as a valuable and versatile tool for biomedical research and future application in the clinic.
Intravital microscopy is here implemented for real-time visualization of intestinal epithelial leakage and observation of epithelial cell shedding events. Leakage of intestinal permeability can be identified by other in vivo noninvasive techniques, such as quantification of orally administration of fluorescent tracers in serum21. However, this technique does not allow the direct observation of shedding performance nor the segregation between para- and trans-cellular permeability. The combination of standard tracer experiments and intravital microscopy represents a suitable approach to: i) identify disturbances in intestinal permeability, and ii) segregate between para- and trans-cellular epithelial permeability. Besides cell shedding, intravital microscopy in combination with in vivo fluorescence labelling enables the study of other cellular and molecular mechanisms (e.g., tight junction redistribution during cell shedding using fluorescent reporter mice22 or interactions between IECs and other cells within the intestinal mucosa23).
The method presented here represents an adaptation of intravital microscopy to enable real-time observation of intestinal mucosa, using confocal laser scanning microscopy (CLSM). Therefore, we use conditional knock-out mice of GGTase (Geranylgeranyltransferase) in intestinal epithelial cells (IECs) in (Pggt1bi&#916;IEC mice), since they suffer from a severe intestinal disease and increased epithelial permeability24. Surgical preparation of the mouse and staining of the intestinal mucosa, as well as appropriate settings used for imaging acquisition and post-acquisition analysis are described. This protocol could enable future studies contributing to the current knowledge about dynamics and kinetics of intestinal epithelial cell shedding. Moreover, the protocol could serve as a basis for various adaptations to study other phenomena occurring at the surface of the intestinal mucosa, and even at other tissues.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Acriflavine hydrochloride</td>
			<td>Sigma Aldrich</td>
			<td>A8251</td>
			<td>1 mg/mL solution in PBS</td>
		</tr>
		<tr>
			<td>Deltaphase isothermal pad</td>
			<td>BrainTree</td>
			<td>B-DP-PAD</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Gemini Cautery System</td>
			<td>BrainTree</td>
			<td>B-GEM-5917</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Ketamin</td>
			<td>WDT</td>
			<td>9089.01.00</td>
			<td></td>
		</tr>
		<tr>
			<td>LAS X</td>
			<td>Leica</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>LSM microscope SP8</td>
			<td>Leica</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Biochrom</td>
			<td>L182</td>
			<td></td>
		</tr>
		<tr>
			<td>Rhodamine B dextran</td>
			<td>Invitrogen</td>
			<td>D1824</td>
			<td>10,000 kDa MW; 2 mg/mL solution</td>
		</tr>
		<tr>
			<td>Standard forceps (Dumont SS)</td>
			<td>Fine Science Tools</td>
			<td>11203-23</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Straight fine scissors</td>
			<td>Fine Science Tools</td>
			<td>14060-10</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Tamoxifen</td>
			<td>Sigma Aldrich</td>
			<td>T5648</td>
			<td>50 mg/mL in ethanol</td>
		</tr>
		<tr>
			<td>Xylazin</td>
			<td>Bayer</td>
			<td>1320422</td>
			<td></td>
		</tr>
	</tbody>
</table>

an intravital microscopy-based approach to assess intestinal permeability and epithelial cell shedding performance

Intravital microscopy of the gut using confocal imaging allows real time observation of epithelial cell shedding and barrier leakage in living animals. Therefore, the intestinal mucosa of anesthetized mice is topically stained with unspecific staining (acriflavine) and a fluorescent tracer (rhodamine-B dextran), mounted on a saline solution-rinsed plate and directly imaged using a confocal microscope. This technique can complement other non-invasive techniques to identify leakage of intestinal permeability, such as transmucosal passage of orally administered tracers. Besides this, the approach presented here allows the direct observation of cell shedding events at real-time. In combination with appropriate fluorescent reporter mice, this approach is suitable for shedding light into cellular and molecular mechanisms controlling intestinal epithelial cell extrusion, as well as to other biological processes. In the last decades, interesting studies using intravital microscopy have contributed to knowledge on endothelial permeability, immune cell gut homing, immune-epithelial communication and invasion of luminal components, among others. Together, the protocol presented here would not only help increase the understanding of mechanisms controlling epithelial cell extrusion, but could also be the basis for the developmental of other approaches to be used as instruments to visualize other highly dynamic cellular process, even in other tissues. Among technical limitations, optical properties of the specific tissue, as well as the selected imaging technology and microscope configuration, would in turn, determine the imaging working distance, and resolution of acquired images.

The protocol presented here describes an intravital microscopy-based approach to visualize intestinal epithelial leakage and observe cell shedding performance in the gut in real-time. Briefly, mice are anesthetized and submitted to surgical preparation in order to expose the surface of the small intestine mucosa. IECs are then stained via topical application of acriflavine; while luminal rhodamine B-dextran is used as tracers to detect transmucosal passage from the lumen to the sub-epithe...

Watch this Scientific Journal Video about An Intravital Microscopy-Based Approach to Assess Intestinal Permeability and Epithelial Cell Shedding Performance at JoVE.com

An Intravital Microscopy-Based Approach to Assess Intestinal Permeability and Epithelial Cell Shedding Performance

Taking advantage of intravital microscopy, the method presented here enables real-time visualization of intestinal epithelial cell shedding in living animals. Therefore, topically stained intestinal mucosa (acriflavine and rhodamineB-dextran) of anesthetized mice is imaged up to single-cell resolution using confocal microscopy.

Intravital microscopy of the gut using confocal imaging allows real time observation of epithelial cell shedding and barrier leakage in living animals. ...

an-intravital-microscopy-based-approach-to-assess-intestinal

Research

JoVE Journal

Immunology and Infection

5.6K Views.  University Hospital of Erlangen. Taking advantage of intravital microscopy, the method presented here enables real-time visualization of intestinal epithelial cell shedding in living animals. Therefore, topically stained intestinal mucosa (acriflavine and rhodamineB-dextran) of anesthetized mice is imaged up to single-cell resolution using confocal microscopy.

Video: An Intravital Microscopy-Based Approach to Assess Intestinal Permeability and Epithelial Cell Shedding Performance

Methods to Assess Beta Cell Death Mediated by Cytotoxic T Lymphocytes

Type 1 diabetes (T1D) is a T cell mediated autoimmune disease. During the pathogenesis, patients become progressively more insulinopenic as 
insulin production is lost, presumably this results from the destruction of pancreatic beta cells by T cells. Understanding the mechanisms of beta cell death during 
the development of T1D will provide insights to generate an effective cure for this disease. Cell-mediated lymphocytotoxicity (CML) assays have historically used the 
radionuclide Chromium 51 (51Cr) to label target cells. These targets are then exposed to effector cells and the release of 51Cr from target 
cells is read as an indication of lymphocyte-mediated cell death. Inhibitors of cell death result in decreased release of 51Cr.
 As effector cells, we used an activated autoreactive clonal population of CD8+ Cytotoxic T lymphocytes (CTL) isolated from a mouse stock transgenic for both the alpha and beta chains of the AI4 T cell receptor (TCR). Activated AI4 T cells were co-cultured with 51Cr labeled target NIT cells for 16 hours, release of 51Cr was recorded to calculate specific lysis 
Mitochondria participate in many important physiological events, such as energy production, regulation of signaling transduction, and apoptosis. The study of beta cell mitochondrial functional changes during the development of T1D is a novel area of research. Using the mitochondrial membrane potential dye Tetramethyl Rhodamine Methyl Ester (TMRM) and confocal microscopic live cell imaging, we monitored mitochondrial membrane potential over time in the beta cell line NIT-1. For imaging studies, effector AI4 T cells were labeled with the fluorescent nuclear staining dye Picogreen. NIT-1 cells and T cells were co-cultured in chambered coverglass and mounted on the microscope stage equipped with a live cell chamber, controlled at 37&deg;C, with 5% CO2, and humidified. During these experiments images were taken of each cluster every 3 minutes for 400 minutes.
Over a course of 400 minutes, we observed the dissipation of mitochondrial membrane potential in NIT-1 cell clusters where AI4 T cells were attached. In the simultaneous control experiment where NIT-1 cells were co-cultured with MHC mis-matched human lymphocyte Jurkat cells, mitochondrial membrane potential remained intact. This technique can be used to observe real-time changes in mitochondrial membrane potential in cells under attack of cytotoxic lymphocytes, cytokines, or other cytotoxic reagents.

Cell-mediated lymphocytotoxicity (CML) assays can be used to test autoreactive responses and study mechanisms of cell death in vitro. However, using live-cell confocal microscopic imaging techniques with fluorescent dyes, the type and kinetics of cell death as well as the pathways utilized can be studied in greater detail.

Type 1 diabetes (T1D) is a T cell mediated autoimmune disease.  During the pathogenesis, patients become progressively more insulinopenic as 
insulin ...

Application of a Mouse Ligated Peyer&#x2019;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed to them and occasionally to pathogens. The gut-associated lymphoid tissue (GALT) play a key role for induction of the mucosal immune response to these microbes1, 2. To initiate the mucosal immune response, the mucosal antigens must be transported from the gut lumen across the epithelial barrier into organized lymphoid follicles such as Peyer's patches. This antigen transcytosis is mediated by specialized epithelial M cells3, 4. M cells are atypical epithelial cells that actively phagocytose macromolecules and microbes. Unlike dendritic cells (DCs) and macrophages, which target antigens to lysosomes for degradation, M cells mainly transcytose the internalized antigens. This vigorous macromolecular transcytosis through M cells delivers antigen to the underlying organized lymphoid follicles and is believed to be essential for initiating antigen-specific mucosal immune responses. However, the molecular mechanisms promoting this antigen uptake by M cells are largely unknown. We have previously reported that glycoprotein 2 (Gp2), specifically expressed on the apical plasma membrane of M cells among enterocytes, serves as a transcytotic receptor for a subset of commensal and pathogenic enterobacteria, including Escherichia coli and Salmonella enterica serovar Typhimurium (S. Typhimurium), by recognizing FimH, a component of type I pili on the bacterial outer membrane 5. Here, we present a method for the application of a mouse Peyer's patch intestinal loop assay to evaluate bacterial uptake by M cells. This method is an improved version of the mouse intestinal loop assay previously described 6, 7. The improved points are as follows: 1. Isoflurane was used as an anesthetic agent. 2. Approximately 1 cm ligated intestinal loop including Peyer's patch was set up. 3. Bacteria taken up by M cells were fluorescently labeled by fluorescence labeling reagent or by overexpressing fluorescent protein such as green fluorescent protein (GFP). 4. M cells in the follicle-associated epithelium covering Peyer's patch were detected by whole-mount immunostainig with anti Gp2 antibody. 5. Fluorescent bacterial transcytosis by M cells were observed by confocal microscopic analysis. The mouse Peyer's patch intestinal loop assay could supply the answer what kind of commensal or pathogenic bacteria transcytosed by M cells, and may lead us to understand the molecular mechanism of how to stimulate mucosal immune system through M cells.

Application of a Mouse Ligated Peyer&amp;#8217;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

M cells in a specialized follicle-associated epithelium covering Peyer&#x2019;s patches play an important role for the mucosal immunosurveillance in gut-associated lymphoid tissue. Here we described the evaluation method for bacterial transcytosis by M cells in vivo. This method provides a method to understand M-cell function in the immune system.

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed ...

A Microplate Assay to Assess Chemical Effects on RBL-2H3 Mast Cell Degranulation: Effects of Triclosan without Use of an Organic Solvent

Mast cells play important roles in allergic disease and immune defense against parasites. Once activated (e.g. by an allergen), they degranulate, a process that results in the exocytosis of allergic mediators. Modulation of mast cell degranulation by drugs and toxicants may have positive or adverse effects on human health. Mast cell function has been dissected in detail with the use of rat basophilic leukemia mast cells (RBL-2H3), a widely accepted model of human mucosal mast cells3-5. Mast cell granule component and the allergic mediator &#946;-hexosaminidase, which is released linearly in tandem with histamine from mast cells6, can easily and reliably be measured through reaction with a fluorogenic substrate, yielding measurable fluorescence intensity in a microplate assay that is amenable to high-throughput studies1. Originally published by Naal et al.1, we have adapted this degranulation assay for the screening of drugs and toxicants and demonstrate its use here.
Triclosan is a broad-spectrum antibacterial agent that is present in many consumer products and has been found to be a therapeutic aid in human allergic skin disease7-11, although the mechanism for this effect is unknown. Here we demonstrate an assay for the effect of triclosan on mast cell degranulation. We recently showed that triclosan strongly affects mast cell function2. In an effort to avoid use of an organic solvent, triclosan is dissolved directly into aqueous buffer with heat and stirring, and resultant concentration is confirmed using UV-Vis spectrophotometry (using &#949;280 = 4,200 L/M/cm)12. This protocol has the potential to be used with a variety of chemicals to determine their effects on mast cell degranulation, and more broadly, their allergic potential.

Mast cell degranulation, the release of allergic mediators, is important in allergy, asthma, and parasite defense. Here we demonstrate techniques1 for assessing effects of drugs and toxicants on degranulation, methodology recently utilized to exhibit the powerful inhibitory effect of antibacterial agent triclosan2.

Mast cells play important roles in allergic disease and immune defense against parasites. Once activated (e.g. by an allergen), they degranulate, a ...

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

Mucosal surfaces serve as protective barriers against pathogenic organisms.&#160;Innate immune responses are activated upon sensing pathogen leading to the infiltration of tissues with migrating inflammatory cells, primarily neutrophils.&#160;This process has the potential to be destructive to tissues if excessive or held in an unresolved state.&#160; Cocultured in vitro models can be utilized to study the unique molecular mechanisms involved in pathogen induced neutrophil trans-epithelial migration.&#160;This type of model provides versatility in experimental design with opportunity for controlled manipulation of the pathogen, epithelial barrier, or neutrophil.&#160;Pathogenic infection of the apical surface of polarized epithelial monolayers grown on permeable transwell filters instigates physiologically relevant basolateral to apical trans-epithelial migration of neutrophils applied to the basolateral surface.&#160;The in vitro model described herein demonstrates the multiple steps necessary for demonstrating neutrophil migration across a polarized lung epithelial monolayer that has been infected with pathogenic P. aeruginosa (PAO1).&#160;Seeding and culturing of permeable transwells with human derived lung epithelial cells is described, along with isolation of neutrophils from whole human blood and culturing of PAO1 and nonpathogenic K12 E. coli (MC1000).&#160; The emigrational process and quantitative analysis of successfully migrated neutrophils that have been mobilized in response to pathogenic infection is shown with representative data, including positive and negative controls.&#160;This in vitro model system can be manipulated and applied to other mucosal surfaces.&#160;Inflammatory responses that involve excessive neutrophil infiltration can be destructive to host tissues and can occur in the absence of pathogenic infections.&#160;A better understanding of the molecular mechanisms that promote neutrophil trans-epithelial migration through experimental manipulation of the in vitro coculture assay system described herein has significant potential to identify novel therapeutic targets for a range of mucosal infectious as well as inflammatory diseases.

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

Neutrophil trans-epithelial migration in response to mucosal bacterial infection contributes to epithelial injury and clinical disease.&#160;An in vitro model has been developed that combines pathogen, human neutrophils, and polarized human epithelial cell layers grown on transwell filters to facilitate investigations towards unraveling the molecular mechanisms orchestrating this phenomenon.

Mucosal surfaces serve as protective barriers against pathogenic organisms.&#160;Innate immune responses are activated upon sensing pathogen leading to ...

Development of an IFN-&#947; ELISpot Assay to Assess Varicella-Zoster Virus-specific Cell-mediated Immunity Following Umbilical Cord Blood Transplantation

Varicella zoster virus (VZV) is a significant cause of morbidity and mortality following umbilical cord blood transplantation (UCBT). For this reason, antiherpetic prophylaxis is administrated systematically to pediatric UCBT recipients to prevent complications associated with VZV infection, but there is no strong, evidence based consensus that defines its optimal duration. Because T cell mediated immunity is responsible for the control of VZV infection, assessing the reconstitution of VZV specific T cell responses following UCBT could provide indications as to whether prophylaxis should be maintained or can be discontinued. To this end, a VZV specific gamma interferon (IFN-&#947;) enzyme-linked immunospot (ELISpot) assay was developed to characterize IFN-&#947; production by T lymphocytes in response to in vitro stimulation with irradiated live attenuated VZV vaccine. This assay provides a rapid, reproducible and sensitive measurement of VZV specific cell mediated immunity suitable for monitoring the reconstitution of VZV specific immunity in a clinical setting and assessing immune responsiveness to VZV antigens. &#160;

Development of an IFN-&amp;#947; ELISpot Assay to Assess Varicella-Zoster Virus-specific Cell-mediated Immunity Following Umbilical Cord Blood Transplantation

Novel generations of functional assays such as gamma interferon (IFN-&#947;) ELISpot, which detect cytokine production at the single cell level and provide both quantitative and qualitative characterization of T cell responses can be used to assess cell-mediated immune responses directed against varicella zoster virus (VZV).

Varicella zoster virus (VZV) is a significant cause of morbidity and mortality following umbilical cord blood transplantation (UCBT). For this reason, ...

A Simple and Efficient Approach to Construct Mutant Vaccinia Virus Vectors

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

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

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

A Robust Pneumonia Model in Immunocompetent Rodents to Evaluate Antibacterial Efficacy against S. pneumoniae, H. influenzae, K. pneumoniae, P. aeruginosa or A. baumannii

Efficacy of candidate antibacterial treatments must be demonstrated in animal models of infection as part of the discovery and development process, preferably in models which mimic the intended clinical indication. A method for inducing robust lung infections in immunocompetent rats and mice is described which allows for the assessment of treatments in a model of serious pneumonia caused by S. pneumoniae, H. influenzae, P. aeruginosa, K. pneumoniae or A. baumannii. Animals are anesthetized, and an agar-based inoculum is deposited deep into the lung via nonsurgical intratracheal intubation. The resulting infection is consistent, reproducible, and stable for at least 48 h and up to 96 h for most isolates. Studies with marketed antibacterials have demonstrated good correlation between in vivo efficacy and in vitro susceptibility, and concordance between pharmacokinetic/pharmacodynamic targets determined in this model and clinically accepted targets has been observed. Although there is an initial time investment when learning the technique, it can be performed quickly and efficiently once proficiency is achieved. Benefits of the model include elimination of the neutropenic requirement, increased robustness and reproducibility, ability to study more pathogens and isolates, improved flexibility in study design and establishment of a challenging infection in an immunocompetent host.

A Robust Pneumonia Model in Immunocompetent Rodents to Evaluate Antibacterial Efficacy against S. pneumoniae, H. influenzae, K. pneumoniae, P. aeruginosa or  A. baumannii

A method for establishing lung infections in immunocompetent rodents is described. With proficiency, this method can be performed quickly and easily to induce stable infection with many isolates of S. pneumoniae, H. influenzae, P. aeruginosa, K. pneumoniae and A. baumannii.

Efficacy of candidate antibacterial treatments must be demonstrated in animal models of infection as part of the discovery and development process, ...

Using Human Induced Pluripotent Stem Cell-derived Intestinal Organoids to Study and Modify Epithelial Cell Protection Against Salmonella and Other Pathogens

The intestinal &#8216;organoid&#8217; (iHO) system, wherein 3-D structures representative of the epithelial lining of the human gut can be produced from human induced pluripotent stem cells (hiPSCs) and maintained in culture, provides an exciting opportunity to facilitate the modeling of the epithelial response to enteric infections. In vivo, intestinal epithelial cells (IECs) play a key role in regulating intestinal homeostasis and may directly inhibit pathogens, although the mechanisms by which this occurs are not fully elucidated. The cytokine interleukin-22 (IL-22) has been shown to play a role in the maintenance and defense of the gut epithelial barrier, including inducing a release of antimicrobial peptides and chemokines in response to infection.
We describe the differentiation of healthy control hiPSCs into iHOs via the addition of specific cytokine combinations to their culture medium before embedding them into a basement membrane matrix-based prointestinal culture system. Once embedded, the iHOs are grown in media supplemented with Noggin, R-spondin-1, epidermal growth factor (EGF), CHIR99021, prostaglandin E2, and Y-27632 dihydrochloride monohydrate. Weekly passages by manual disruption of the iHO ultrastructure lead to the formation of budded iHOs, with some exhibiting a crypt/villus structure. All iHOs demonstrate a differentiated epithelium consisting of goblet cells, enteroendocrine cells, Paneth cells, and polarized enterocytes, which can be confirmed via immunostaining for specific markers of each cell subset, transmission electron microscopy (TEM), and quantitative PCR (qPCR). To model infection, Salmonella enterica serovar Typhimurium SL1344 are microinjected into the lumen of the iHOs and incubated for 90 min at 37 &#176;C, and a modified gentamicin protection assay is performed to identify the levels of intracellular bacterial invasion. Some iHOs are also pretreated with recombinant human IL-22 (rhIL-22) prior to infection to establish whether this cytokine is protective against Salmonella infection.

Using Human Induced Pluripotent Stem Cell-derived Intestinal Organoids to Study and Modify Epithelial Cell Protection Against Salmonella and Other Pathogens

Human induced pluripotent stem cell (hiPSC)-derived intestinal organoids offer exciting opportunities to model enteric diseases in vitro. We demonstrate the differentiation of hiPSCs into intestinal organoids (iHOs), the stimulation of these iHOs with cytokines, and the microinjection of Salmonella Typhimurium into the iHO lumen, enabling the study of an epithelial invasion by this pathogen.

The intestinal &#8216;organoid&#8217; (iHO) system, wherein 3-D structures representative of the epithelial lining of the human gut can be produced from ...

Functional Assessment of Intestinal Permeability and Neutrophil Transepithelial Migration in Mice using a Standardized Intestinal Loop Model

The intestinal mucosa is lined by a single layer of epithelial cells that forms a dynamic barrier allowing paracellular transport of nutrients and water while preventing passage of luminal bacteria and exogenous substances. A breach of this layer results in increased permeability to luminal contents and recruitment of immune cells, both of which are hallmarks of pathologic states in the gut including inflammatory bowel disease (IBD). 
Mechanisms regulating epithelial barrier function and transepithelial migration (TEpM) of polymorphonuclear neutrophils (PMN) are incompletely understood due to the lack of experimental in vivo methods allowing quantitative analyses. Here, we describe a robust murine experimental model that employs an exteriorized intestinal segment of either ileum or proximal colon. The exteriorized intestinal loop (iLoop) is fully vascularized and offers physiological advantages over ex vivo chamber-based approaches commonly used to study permeability and PMN migration across epithelial cell monolayers.
We demonstrate two applications of this model in detail: (1) quantitative measurement of intestinal permeability through detection of fluorescence-labeled dextrans in serum after intraluminal injection, (2) quantitative assessment of migrated PMN across the intestinal epithelium into the gut lumen after intraluminal introduction of chemoattractants. We demonstrate feasibility of this model and provide results utilizing the iLoop in mice lacking the epithelial tight junction-associated protein JAM-A compared to controls. JAM-A has been shown to regulate epithelial barrier function as well as PMN TEpM during inflammatory responses. Our results using the iLoop confirm previous studies and highlight the importance of JAM-A in regulation of intestinal permeability and PMN TEpM in vivo during homeostasis and disease. 
The iLoop model provides a highly standardized method for reproducible in vivo studies of intestinal homeostasis and inflammation and will significantly enhance understanding of intestinal barrier function and mucosal inflammation in diseases such as IBD.

Dysregulated intestinal epithelial barrier function and immune responses are hallmarks of inflammatory bowel disease that remain poorly investigated due to a lack of physiological models. Here, we describe a mouse intestinal loop model that employs a well-vascularized and exteriorized bowel segment to study mucosal permeability and leukocyte recruitment in vivo.

The intestinal mucosa is lined by a single layer of epithelial cells that forms a dynamic barrier allowing paracellular transport of nutrients and water ...

Real-Time Quantification of Reactive Oxygen Species in Neutrophils Infected with Meningitic Escherichia Coli

Escherichia coli (E. coli) is the most common Gram-negative bacteria causing neonatal meningitis. The occurrence of bacteremia and bacterial penetration through the blood-brain barrier are indispensable steps for the development of E. coli meningitis. Reactive oxygen species (ROS) represent the major bactericidal mechanisms of neutrophils to destroy the invaded pathogens. In this protocol, the time-dependent intracellular ROS production in neutrophils infected with meningitic E. coli was quantified using fluorescent ROS probes detected by a real-time fluorescence microplate reader. This method may also be applied to the assessment of ROS production in mammalian cells during pathogen-host interactions.

Real-Time Quantification of Reactive Oxygen Species in Neutrophils Infected with Meningitic Escherichia Coli

Escherichia coli is the leading cause of neonatal Gram-negative bacterial meningitis. During the bacterial infection, reactive oxygen species produced by neutrophils play a major bactericidal role. Here we introduce a method to detect the reactive oxygen species in neutrophils in response to meningitis E. coli.

Escherichia coli (E. coli) is the most common Gram-negative bacteria causing neonatal meningitis. The occurrence of bacteremia and bacterial penetration ...