This work was supported by the Canadian Institutes of Health Research (MSH-95368, MOP-89773 and MOP-106623 to C.Z.) and the Canada Foundation for Innovation. S.C.M. is the recipient of a CIHR/Canadian Association of Gastroenterology postdoctoral fellowship. C.Z. is a CIHR New Investigator.

1. Propagating Trichuris muris eggs
<ol>
<li>To generate new batches of Trichuris muris eggs, infect 20-30 immunodeficient mice (eg. NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ 
(NSG) or 129S6/SvEvTac-Rag2tm1Fwa (RAG2-/-)) or genetically susceptible mice (eg. AKR/J) that are 6-8 weeks old with approximately 300 Trichuris 
muris eggs by oral gavage.</li> 
<li>After 32&#x2013;35 days sacrifice the mice by CO2 asphyxiation.</li>
<li>Expose the ventral side of the mouse and wet the abdomen with 70% ethanol.</li>
<li>Using forceps grasp the abdominal skin and make a small incision using scissors. Cut the skin to expose the abdominal contents.</li>
<li>Identify the cecum and the proximal large intestine, and remove them.</li>
<li>Place the cecum and proximal large intestine in a 10 cm petri dish containing Dulbecco's Modified Eagle Medium (DMEM) with 500 U/ml penicillin and 500 &mu;g/ml 
streptomycin (P/S). Cut the cecum open to expose the lumen and the worms. Using forceps place cecum in a beaker of phosphate buffered saline (PBS) and shake gently to remove feces.</li>
<li>Return cecum to petri dish and using smooth curved forceps (Roboz RS-5047) gently pull out worms and place them in a well of a 6 well plate containing 5 mls of DMEM + P/S.</li>
<li>Repeat this process for all of the mice.</li>
<li>Put 6 well plate in a Tupperware container with a damp paper towel to maintain humidity and incubate at 37&deg;C for 4 hrs.</li>
<li>After 4 hrs remove worms using smooth curved forceps and split them into 2 new wells of the 6 well plate containing 5mL DMEM + P/S. Harvest the contents of the original well and place in a 15 ml falcon tube. Spin tube at 3000 rpm and remove and reserve supernatant as 4 hr antigen (can be used for cell restimulation after PBS dialysis, where typical protein yields are 10-20 mg per 20 mice) and resuspend egg-containing pellet in autoclaved tap water.</li>
<li>Put the 6 well plate back at 37&deg;C overnight. Remove and dispose of the worms. Harvest the contents of the wells into a falcon tube and spin at 3000 rpm for 5 min. Remove supernatant and reserve as overnight antigen (can be used for Trichuris Ag specific antibody isotype ELISAs after PBS dialysis, where typical protein yields are 10-20 mg per 20 mice) and resuspend egg-containing pellet in autoclaved tap water.</li>
<li>Combine 4 hr and overnight eggs and filter the eggs through the 70&mu;m strainer to remove any leftover worms then transfer into a 75 cm2 flask. Keep 
flask in a dark place covered in foil for 6 weeks at room temperature (RT).</li>
<li>Wash eggs in autoclaved tap water and resuspend at 50 viable eggs in 50 &mu;l. To do this, centrifuge eggs at 3000 rpm for 5 min and resuspend in &tilde;50ml sterile tap water. Remove 50 &mu;l eggs and place on a glass slide. Using the 40x magnification count the number of viable embryonated eggs in the 50 &mu;l. Dilute eggs to give 50 viable eggs in 50 &mu;l. For images of viable eggs please see the study by Kopper and Mansfield16.</li>
</ol>
2. Acute infection of experimental mice with Trichuris muris eggs (200 eggs/mouse)
<ol>
<li>Thoroughly resuspend eggs and remove enough volume to treat the number of mice X 2 (eg. for 10 mice take 200 &mu;l x 10 mice x 2 = 4 mls of eggs) and place them 
in a 14 ml snap cap tube.</li>
<li>Invert the 14 ml snap cap tube to mix. Bring up 200 &mu;l of eggs into 1 ml syringe on an appropriate size feeding needle (for mice weighing 25 grams we used 18 gauge 1 1/2" needles). In one hand scruff the mouse and 
using the other hand administer eggs by oral gavage .</li>
<li>Wait 21 days.</li> 
</ol>
3. Experimental harvest
<ol>
<li>On day 21 sacrifice the mice using CO2.</li>
<li>Collect blood from mice by cardiac puncture, in a non-heparinized syringe, and store at 4&deg;C. The following day isolate serum and freeze at -20&deg;C. Serum can be used for detection of Trichuris-specific IgGs and IgE by ELISA.</li>
<li>Expose the ventral side of the mouse and wet the abdomen with 70% ethanol.</li>
<li>Using forceps grasp the abdominal skin and make a small incision using scissors. Cut the skin and underlying membrane to expose the abdominal contents.</li> 
<li>Identify the small intestine, cecum and colon. Grab the cecum with forceps and pull out of abdominal cavity. With another pair of forceps push small intestine over to the right exposing the mesenteric lymph nodes (mLNs). Remove the mLNs being careful not to cut into the intestines and place in a 15 ml falcon containing 2 mls of media. If desired these can be processed and restimulated in vitro (4x106/ml in 1 ml in 24 well plates with 1 &mu;g/ml &alpha;CD3/CD28 or with 50&mu;g/ml 4h Trichuris Ag) for 72 hrs for cytokine analysis by ELISA and intracellular flow cytometry.</li>
<li>Cut out the cecum and colon.</li> 
<li>Using forceps, push out fecal pellets from distal colon and place in a 2 ml Axygen tube. Freeze at -20&deg;C. Fecal pellets can be analyzed by western blot for expression of resistin-like molecule-&beta; (RELM-&beta;).</li>
<li>Remove the tip of the cecum (&tilde;0.5 cm). To remove feces hold cecum tip with forceps and flush with PBS using a 3 ml syringe and 22-gauge needle in a petri dish. Place in fresh 4% paraformaldehyde in a 5 ml Falcon snap cap tube. Store at 4&deg;C. These can be paraffin-embedded, sectioned and mounted on slides for histological analysis.</li>
<li>Remove a piece of proximal colon (&tilde; 1 cm) for RNA. Put sample into 2 ml Axygen tube with 500 &mu;l RNAlater. Store at 4&deg;C. These samples can be analyzed for mRNA expression by qPCR.</li> 
<li>Place the rest of the cecum in a labeled petri dish and freeze at -20&deg;C.</li>
</ol>
4. Enumeration of Trichuris muris worms in the cecum
<ol>
<li>Remove cecum from freezer and place in a petri dish containing &tilde;5 ml water.</li>
<li>Using scissors, cut open cecum to expose the lumen.</li>
<li>Hold the cecum with forceps and vigorously shake the cecum to remove the majority of the feces.</li>
<li>Transfer cecum to another petri dish containing water and using smooth curved forceps gently scrape off the IECs the underlying tissue.</li>
<li>Transfer cecum to a new petri dish containing water and vigorously scrape remaining tissue with forceps, ripping the tissue into small pieces.</li> 
<li>Use a dissecting microscope to count all of the worms in all 3 of the petri dishes. As you find a worm remove it from the dish to ensure the same worm isn&#x2019;t counted twice. If worms are damaged (i.e. broken in half) count only thick or thin ends to get an accurate count.</li>
</ol>
5. Representative Results
The Trichuris muris infection model allows researchers to quantify parasite burden and systemic immune responses, as well as to histologically examine the inflammatory response within the distal cecum. When the lumen of the harvested cecum is exposed, Trichuris worms can be enumerated using a dissecting microscope (Figure 1A-B). Parameters of immunity to Trichuris also include antibody isotype switching to IgG1 (associated with Th2-type immunity) in resistant animals, and IgG2a (associated with Th1-type immunity) in susceptible animals (Figure 1C). Expression of the goblet cell-specific protein resistin-like molecule-&beta; (RELM-&beta;) is associated with clearance of Trichuris and is detectable by Western blot analysis (Figure 2). The distal portion of the cecum can be stained with Periodic Acid-Schiff (PAS) to assess goblet cell responses and the extent of intestinal inflammation in response to infection (Figure 3).
<img src="/files/ftp_upload/2774/2774fig1.jpg" alt="Figure 1"/> Figure 1. Quantification of immunity to Trichuris 
muris. (A) Resistant (B6) mice expel Trichuris muris by day 21 post-infection. By contrast, susceptible (AKR/J) mice become chronically infected 
with Trichuris, resulting in parasite establishment. Each bar represents the mean &plusmn; SEM of 4-8 animals per group. (B) Worms are counted using a 
dissecting microscope. Top panel shows worms in isolation, bottom panel shows worms in the presence of IECs and feces. (C) Trichuris-specific IgG2a 
titres are determined by ELISA of diluted serum (1:2 dilution, starting at 1:20 dilution and ending at 1:2560) on plates coated with excretory Trichuris 
antigen (O/N Ag, 5 &mu;g/ml). Susceptible AKR/J mice have notably higher levels of Trichuris-specific serum IgG2a than resistant B6 mice.
<img src="/files/ftp_upload/2774/2774fig2.jpg" alt="Figure 2"/> 
Figure 2. Clearance of Trichuris muris is associated with expression of the goblet cell-specific protein RELM-&beta;7. 
RELM-&beta; production can be detected from fecal pellets from Trichuris-infected resistant (B6), but not susceptible (AKR/J) mice by western blot 
analysis. Each lane is representative of a single animal, (N)=Naive, (Tm)=21-day Trichuris infected mice.
<img src="/files/ftp_upload/2774/2774fig3.jpg" alt="Figure 3"/> Figure 3. Histological examination of the distal cecum 
following Trichuris infection. 5-7 &mu;m sections of the distal cecum stained with PAS. Goblet cell hyperplasia and mucus production are 
evident in resistant (B6) but not susceptible (AKR/J) mice following Trichuris infection.

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	<li>Cliffe, L. J., Grencis, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Trichuris+muris+system:+a+paradigm+of+resistance+and+susceptibility+to+intestinal+nematode+infection.">The Trichuris muris system: a paradigm of resistance and susceptibility to intestinal nematode infection.</a> Adv. Parasitol. 57, 255-307 (2004).</li><li>Else, K. J., Finkelman, F. D., Maliszewski, C. R., Grencis, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytokine-mediated+regulation+of+chronic+intestinal+helminth+infection.">Cytokine-mediated regulation of chronic intestinal helminth infection.</a> J. Exp. Med. 179, 347-351 (1994).</li><li>Bancroft, A. J., Grencis, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Th1+and+Th2+cells+and+immunity+to+intestinal+helminths.">Th1 and Th2 cells and immunity to intestinal helminths.</a> Chem. Immunol. 71, 192-208 (1998).</li><li>Bancroft, A. J., McKenzie, A. N., Grencis, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+critical+role+for+IL-13+in+resistance+to+intestinal+nematode+infection.">A critical role for IL-13 in resistance to intestinal nematode infection.</a> J. Immunol. 160, 3453-3461 (1998).</li><li>Helmby, H., Takeda, K., Akira, S., Grencis, R. 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Experiments on animals were performed in accordance with the guidelines and regulations set forth by The University of British Columbia.

This protocol details a standard high dose acute Trichuris muris infection which can be modified as required by the investigator. For example, mice can be sacrificed and tissues harvested on different days. To determine that the mice have successfully established full worm burden they can be sacrificed on day 14, at which point all mice should carry a burden of approximately 200 worms. Mice can also be infected for 32 days where any worm detected will have reached maturity and would remain with host for the d...

<table border="1">
	<tbody>
 <tr>
 	<th>Name of the reagent</th>
 <th>Company</th>
 <th>Catalogue number</th>
 <th>Comments</th>
 </tr>
 <tr>
 	<td>Animal Feeding Needles (18 x 1&frac12;")</td>
 <td>Popper</td>
 <td>7912</td>
 <td>&nbsp;</td>
 </tr>
		<tr>
 	<td>Smooth curved Forceps</td>
 <td>Roboz</td>
 <td>RS-5047</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>DMEM</td>
 <td>Gibco</td>
 <td>11965</td>
 <td>&nbsp;</td>
 </tr>
		<tr>
 	<td>NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG)</td>
 <td>Jackson Laboratories</td>
 <td>005557</td>
 <td>These are the mice we used, however, any immunodeficient mice or susceptible strain should work.</td>
 </tr>
 <tr>
 	<td>RNAlater</td>
 <td>Qiagen</td>
 <td>76104</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>2 ml tubes</td>
 <td>Axygen</td>
 <td>MCT-200-C</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>15 ml tubes</td>
 <td>Falcon</td>
 <td>352096</td>
 <td>&nbsp;</td>
 </tr><tr>
 	<td>6 well plates</td>
 <td>Falcon</td>
 <td>353046</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Paraformaldehyde</td>
 <td>Electron Microscopy Science</td>
 <td>15710</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>&alpha;-RELM&beta; antibody</td>
 <td>PeproTech Inc</td>
 <td>0694270Rb</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>

trichuris muris infection: a model of type 2 immunity and inflammation in the gut

Trichuris muris is a natural pathogen of mice and is biologically and antigenically similar to species of Trichuris that 
infect humans and livestock1. Infective eggs are given by oral gavage, hatch in the distal small intestine, invade the intestinal epithelial cells (IECs) 
that line the crypts of the cecum and proximal colon and upon maturation the worms release eggs into the environment1. This model is a powerful tool to 
examine factors that control CD4+ T helper (Th) cell activation as well as changes in the intestinal epithelium. The immune response that occurs in 
resistant inbred strains, such as C57BL/6 and BALB/c, is characterized by Th2 polarized cytokines (IL-4, IL-5 and IL-13) and expulsion of worms while Th1-associated 
cytokines (IL-12, IL-18, IFN-&gamma;) promote chronic infections in genetically susceptible AKR/J mice2-6. Th2 cytokines promote physiological changes in 
the intestinal microenvironment including rapid turnover of IECs, goblet cell differentiation, recruitment and changes in epithelial permeability and smooth muscle 
contraction, all of which have been implicated in worm expulsion7-15. Here we detail a protocol for propagating Trichuris muris eggs which can 
be used in subsequent experiments. We also provide a sample experimental harvest with suggestions for post-infection analysis. Overall, this protocol will provide 
researchers with the basic tools to perform a Trichuris muris mouse infection model which can be used to address questions pertaining to Th proclivity in 
the gastrointestinal tract as well as immune effector functions of IECs.

Watch this Scientific Journal Video about Trichuris muris Infection: A Model of Type 2 Immunity and Inflammation in the Gut at JoVE.com

Trichuris muris Infection: A Model of Type 2 Immunity and Inflammation in the Gut

Trichuris muris infection is an intestinal model of Th2 immunity where resistant mice generate a protective Th2 response and susceptible mice generate a pathological Th1 response.

Trichuris muris Infection: A Model of Type 2 Immunity and Inflammation in the Gut

Trichuris muris is a natural pathogen of mice and is biologically and antigenically similar to species of Trichuris that 
infect humans and livestock1. ...

trichuris-muris-infection-model-type-2-immunity-inflammation

Research

JoVE Journal

Immunology and Infection

16.2K Görüntüleme.  University of British Columbia. Trichuris muris enfeksiyon dayanıklı farelerde koruyucu bir Th2 yanıtı oluşturmak ve duyarlı fareler patolojik bir Th1 yanıtı Th2 dokunulmazlık bağırsak modelidir.

Video: Trichuris muris Infection: A Model of Type 2 Immunity and Inflammation in the Gut

Capsular Serotyping of Streptococcus pneumoniae by Latex Agglutination

Latex agglutination reagents are widely used in microbial diagnosis, identification and serotyping. Streptococcus pneumoniae (the pneumococcus) is a major cause of morbidity and mortality world-wide. Current vaccines target the pneumococcal capsule, and there are over 90 capsular serotypes. Serotyping pneumococcal isolates is therefore important for assessing the impact of vaccination programs and for epidemiological purposes. The World Health Organization has recommended latex agglutination as an alternative method to the &#8216;gold standard&#8217; Quellung test for serotyping pneumococci. Latex agglutination is a relatively simple, quick and inexpensive method; and is therefore suitable for resource-poor settings as well as laboratories with high-volume workloads. Latex agglutination reagents can be prepared in-house utilizing commercially-sourced antibodies that are passively attached to latex particles. This manuscript describes a method of production and quality control of latex agglutination reagents, and details a sequential testing approach which is time- and cost-effective. 
This method of production and quality control may also be suitable for other testing purposes.

Capsular Serotyping of Streptococcus pneumoniae by Latex Agglutination

Latex agglutination testing is a simple, rapid and inexpensive method for serotyping Streptococcus pneumoniae, and has also been widely applied in diagnostic microbiology. This manuscript describes the in-house production of latex agglutination reagents, quality control procedures and the application of this technique to pneumococcal serotyping.

Latex agglutination reagents are widely used in microbial diagnosis, identification and serotyping. Streptococcus pneumoniae (the pneumococcus) is a major ...

Isolation, Characterization and Functional Examination of the Gingival Immune Cell Network

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident immune cell populations in the oral mucosa and gingiva. We have established a technique for the isolation and study of immune cells from murine gingival tissues, an area of constant microbial exposure and a vulnerable site to a common inflammatory disease, periodontitis. Our protocol allows for a detailed phenotypic characterization of the immune cell populations resident in the gingiva, even at steady state. Our procedure also yields sufficient cells with high viability for use in functional studies, such as the assessment of cytokine secretion ex vivo. This combination of phenotypic and functional characterization of the gingival immune cell network should aid towards investigating the mechanisms involved in oral immunity and periodontal homeostasis, but will also advance our understanding of the mechanisms involved in local immunopathology.

We have established a technique for the isolation, phenotypic characterization and functional analysis of immune cells from murine gingiva.

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

Applying Fluorescence Resonance Energy Transfer (FRET) to Examine Effector Translocation Efficiency by Coxiella burnetii during siRNA Silencing

Coxiella burnetii, the causative agent of Q fever, is an intracellular pathogen that relies on a Type IV Dot/Icm Secretion System to establish a replicative niche. A cohort of effectors are translocated through this system into the host cell to manipulate host processes and allow the establishment of a unique lysosome-derived vacuole for replication. The method presented here involves the combination of two well-established techniques: specific gene silencing using siRNA and measurement of effector translocation using a FRET-based substrate that relies on &#946;-lactamase activity. Applying these two approaches, we can begin to understand the role of host factors in bacterial secretion system function and effector translocation. In this study we examined the role of Rab5A and Rab7A, both important regulators of the endocytic trafficking pathway. We demonstrate that silencing the expression of either protein results in a decrease in effector translocation efficiency. These methods can be easily modified to examine other intracellular and extracellular pathogens that also utilize secretion systems. In this way, a global picture of host factors involved in bacterial effector translocation may be revealed.

Applying Fluorescence Resonance Energy Transfer FRET to Examine Effector Translocation Efficiency by Coxiella burnetii during siRNA Silencing

Investigating the interactions between bacterial pathogens and their hosts is an important area of biological research. Here, we describe the necessary techniques to measure effector translocation by Coxiella burnetii during siRNA gene silencing using BlaM substrate.

Coxiella burnetii, the causative agent of Q fever, is an intracellular pathogen that relies on a Type IV Dot/Icm Secretion System to establish a ...

A Non-invasive and Technically Non-intensive Method for Induction and Phenotyping of Experimental Bacterial Pneumonia in Mice

Although community-acquired pneumonia remains a major public health problem, murine models of bacterial pneumonia have recently facilitated significant preclinical advances in our understanding of the underlying cellular and molecular pathogenesis. In vivo mouse models capture the integrated physiology and resilience of the host defense response in a manner not revealed by alternative, simplified ex vivo approaches. Several methods have been described in the literature for intrapulmonary inoculation of bacteria in mice, including aerosolization, intranasal delivery, peroral endotracheal cannulation under &#39;blind&#39; and visualized conditions, and transcutaneous endotracheal cannulation. All methods have relative merits and limitations. Herein, we describe in detail a non-invasive, technically non-intensive, inexpensive, and rapid method for intratracheal delivery of bacteria that involves aspiration (i.e., inhalation) by the mouse of an infectious inoculum pipetted into the oropharynx while under general anesthesia. This method can be used for pulmonary delivery of a wide variety of non-caustic biological and chemical agents, and is relatively easy to learn, even for laboratories with minimal prior experience with pulmonary procedures. In addition to describing the aspiration pneumonia method, we also provide step-by-step procedures for assaying the subsequent in vivo pulmonary innate immune response of the mouse, in particular, methods for quantifying bacterial clearance and the cellular immune response of the infected airway. This integrated and simple approach to pneumonia assessment allows for rapid and robust evaluation of the effect of genetic and environmental manipulations upon pulmonary innate immunity.

Several methods have been described in the literature for modeling bacterial pneumonia in mice. Herein, we describe a non-invasive, inexpensive, rapid method for inducing pneumonia via aspiration (i.e., inhalation) of a bacterial inoculum pipetted into the oropharynx. Downstream methods for assessment of the pulmonary innate immune response are also detailed.

Although community-acquired pneumonia remains a major public health problem, murine models of bacterial pneumonia have recently facilitated significant ...

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

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

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

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

Fluorescence-based Neuraminidase Inhibition Assay to Assess the Susceptibility of Influenza Viruses to The Neuraminidase Inhibitor Class of Antivirals

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against currently circulating strains. In addition to their use in treating seasonal influenza, the NA inhibitors have been stockpiled by a number of countries for use in the event of a pandemic. It is therefore important to monitor the susceptibility of circulating influenza viruses to this class of antivirals. There are different types of assays that can be used to assess the susceptibility of influenza viruses to the NA inhibitors, but the enzyme inhibition assays using either a fluorescent substrate or a chemiluminescent substrate are the most widely used and recommended. This protocol describes the use of a fluorescence-based assay to assess influenza virus susceptibility to NA inhibitors. The assay is based on the NA enzyme cleaving the 2&#8242;-(4-Methylumbelliferyl)-&#945;-D-N-acetylneuraminic acid (MUNANA) substrate to release the fluorescent product 4-methylumbelliferone (4-MU). Therefore, the inhibitory effect of an NA inhibitor on the influenza virus NA is determined based on the concentration of the NA inhibitor that is required to reduce 50% of the NA activity, given as an IC50 value.

We describe the use of a phenotypic fluorescence-based neuraminidase inhibition assay to assess the susceptibility of influenza A and B viruses to the neuraminidase inhibitor class of antivirals.

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against ...

High-throughput Measurement of Plasma Membrane Resealing Efficiency in Mammalian Cells

In their physiological environment, mammalian cells are often subjected to mechanical and biochemical stresses that result in plasma membrane damage. In response to these damages, complex molecular machineries rapidly reseal the plasma membrane to restore its barrier function and maintain cell survival. Despite 60 years of research in this field, we still lack a thorough understanding of the cell resealing machinery. With the goal of identifying cellular components that control plasma membrane resealing or drugs that can improve resealing, we have developed a fluorescence-based high-throughput assay that measures the plasma membrane resealing efficiency in mammalian cells cultured in microplates. As a model system for plasma membrane damage, cells are exposed to the bacterial pore-forming toxin listeriolysin O (LLO), which forms large 30-50 nm diameter proteinaceous pores in cholesterol-containing membranes. The use of a temperature-controlled multi-mode microplate reader allows for rapid and sensitive spectrofluorometric measurements in combination with brightfield and fluorescence microscopy imaging of living cells. Kinetic analysis of the fluorescence intensity emitted by a membrane impermeant nucleic acid-binding fluorochrome reflects the extent of membrane wounding and resealing at the cell population level, allowing for the calculation of the cell resealing efficiency. Fluorescence microscopy imaging allows for the enumeration of cells, which constitutively express a fluorescent chimera of the nuclear protein histone 2B, in each well of the microplate to account for potential variations in their number and allows for eventual identification of distinct cell populations. This high-throughput assay is a powerful tool expected to expand our understanding of membrane repair mechanisms via screening for host genes or exogenously added compounds that control plasma membrane resealing.

Here we describe a high-throughput fluorescence-based assay that measures the plasma membrane resealing efficiency through fluorometric and imaging analyses in living cells. This assay can be used for screening drugs or target genes that regulate plasma membrane resealing in mammalian cells.

In their physiological environment, mammalian cells are often subjected to mechanical and biochemical stresses that result in plasma membrane damage. In ...

Effects of Taste Signaling Protein Abolishment on Gut Inflammation in an Inflammatory Bowel Disease Mouse Model

Inflammatory bowel disease (IBD) is one of the immune-related gastrointestinal disorders, including ulcerative colitis and Crohn's disease, that affects the life quality of millions of people worldwide. IBD symptoms include abdominal pain, diarrhea, and rectal bleeding, which may result from the interactions among gut microbiota, food components, intestinal epithelial cells, and immune cells. It is of particular importance to assess how each key gene expressed in intestinal epithelial and immune cells affects inflammation in the colon. G protein-coupled taste receptors, including G protein subunit &#945;-gustducin and other signaling proteins, have been found in the intestines. Here, we use &#945;-gustducin as a representative and describe a dextran sulfate sodium (DSS)-induced IBD model to evaluate the effect of gustatory gene mutations on gut mucosal immunity and inflammation. This method combines gene knockout technology with the chemically induced IBD model, and thus can be applied to assess the outcome of gustatory gene nullification as well as other genes that may exuberate or dampen the DSS-induced immune response in the colon. Mutant mice are administered with DSS for a certain period during which their body weight, stool, and rectal bleeding are monitored and recorded. At different timepoints during administration, some mice are euthanized, then the sizes and weights of their spleens and colons are measured and gut tissues are collected and processed for histological and gene expression analyses. The data show that the &#945;-gustducin knockout results in excessive weight loss, diarrhea, intestinal bleeding, tissue damage, and inflammation vs. wild-type mice. Since the severity of induced inflammation is affected by mouse strains, housing environment, and diet, optimization of DSS concentration and administration duration in a pilot experiment is particularly important. By adjusting these factors, this method can be applied to assess both anti- and pro-inflammatory effects.

Here we present a protocol to investigate the effect of the nullification of gustation-related genes on immune responses in a dextran sulfate sodium (DSS)-induced inflammatory bowel disease (IBD) mouse model.

Inflammatory bowel disease (IBD) is one of the immune-related gastrointestinal disorders, including ulcerative colitis and Crohn's disease, that affects ...

The Galleria mellonella Waxworm Infection Model for Disseminated Candidiasis

Candida species are common fungal commensals of humans colonizing the skin, mucosal surfaces, and gastrointestinal tract. Under certain conditions, Candida can overgrow their natural niches resulting in debilitating mucosal infections as well as life-threatening systemic infections, which are a major focus of investigation due to their associated high mortality rates. Animal models of disseminated infection exist for studying disease progression and dissecting the characteristics of Candida pathogenicity. Of these, the Galleria mellonella waxworm infection model provides a cost-effective experimental tool for high-throughput investigations of systemic virulence. Many other bacterial and eukaryotic infectious agents have been effectively studied in G. mellonella to understand pathogenicity, making it a widely accepted model system. Yet, variation in the method used to infect G. mellonella can alter phenotypic outcomes and complicate interpretation of the results. Here, we outline the benefits and drawbacks of the waxworm model to study systemic Candida pathogenesis and detail an approach to improve reproducibility. Our results highlight the range of mortality kinetics in G. mellonella and describe the variables which can modulate these kinetics. Ultimately, this method stands as an ethical, rapid, and cost-effective approach to study virulence in a model of disseminated candidiasis.

The Galleria mellonella Waxworm Infection Model for Disseminated Candidiasis

Galleria mellonella serves as an invertebrate model for disseminated candidiasis. Here, we detail the infection protocol and provide supporting data for the model&#39;s effectiveness.

Candida species are common fungal commensals of humans colonizing the skin, mucosal surfaces, and gastrointestinal tract. Under certain conditions, ...