This work was supported by NIH grants P01 HL108801, R01-HL60234, R01-HL55330, R01-HL079904, to A. M. K. Choi. S. Ryter received salary support from the Lovelace Respiratory Research Institute.

Note:&#160; The Institutional Animal Care and Use Committee at Brigham and Women&#39;s Hospital/ Harvard Medical School Area approved the following procedures.
1. Cecal Ligation and Puncture
Use mice of the same background (C57Bl/6), male, 8-10 weeks old. Female mice are more resistant than males against sepsis-induced lethality. Mice older than 8 weeks produce less variable results than younger mice in terms of survival after CLP. Approximately N=10 mice per compared group should be used for survival analysis. N=3-5 mice is adequate for autophagy assays.
<ol>
	<li>Use sterile small surgical instruments (namely scalpel, scissors and blunt anatomical forceps), which should be appropriate for rodent surgery.</li>
	<li>Prepare anesthesia mixture: To 7.95 ml of PBS add 150 &#956;l of Xylazine (AnaSed injection&#160;100 mg/ml) and 700 &#956;l of Ketamine (Ketaset&#160;CIII, 100 mg/ml).</li>
	<li>To ensure aseptic conditions during the procedure, wear gloves, face mask and surgical gown.</li>
	<li>Weigh animals. Anesthetize them by injecting intraperitonally (i.p.) the mixture mentioned in step 3 in a dosage of &#956;l&#160;(10x body weight in g) . For example, for a mouse weighing 22 g, administer 10x22= 220 &#956;l of the anesthetic mixture. Thus, the final doses of Xylazine and Ketamine are 17 and 80 mg/kg body weight, respectively. Ensure that anesthesia is adequate; no flexion of extremity should be produced after toe pinch. Note: Use ophthalmic&#160;ointment on eyes to prevent dryness while under anesthesia.</li>
	<li>Place animals onto a clean work surface on their backs. Tails and hind paws are oriented towards the investigator. Disinfect the abdominal skin using alcohol (70% EtOH solution). Note: Surgical area should be completely shaved, to avoid contamination of the wound through contact with fur.&#160;Also, optimally, a circulating warm water pad should be used to maintain normothermia of anesthetized mice during the surgery.</li>
	<li>By using a scalpel, make a small longitudinal skin incision parallel and approximately 1 cm left to midline. Do not penetrate yet into the peritoneal cavity. Use small scissors to extend the initial incision.</li>
	<li>Identify abdominal muscles and dissect them to gain access into the peritoneal cavity.</li>
	<li>By using blunt anatomical forceps, identify the cecum and move it out of the peritoneal cavity. Be careful not to damage mesenterial blood vessels (this could lead to lethal hemorrhage).</li>
	<li>Ligate 60% of the cecum to achieve a mid-grade sepsis. Ligating equal to or more than 75% of the cecum generally results in high-grade sepsis, whereas ligating equal to or less than 25% of the cecum results in low-grade sepsis. Be careful not to ligate the ileocecal valve (this could block the intestinal continuity).</li>
	<li>By using a 21&#160;G needle, perforate the cecum by one through-and-through puncture (two holes) near to the ligation. The direction of the perforation should be from the mesenteric to the anti-mesenteric side of the cecum. Be careful not to puncture mesenterial blood vessels.</li>
	<li>Remove the needle. Gently squeeze the ligated cecum to confirm patency through the two holes; only a small amount of feces should be extruded through them.</li>
	<li>Move the ligated and punctured cecum into the peritoneal cavity. Note: For sham mice, skip steps 1.10-1.12.</li>
	<li>Use silk surgical sutures 6-0 with cutting needle to close abdominal musculature. Use silk surgical sutures 6-0 with cutting needle to close abdominal skin.</li>
	<li>Inject 1 ml&#160;prewarmed (37 &#176;C) normal saline i.p. to replace heat and hydration lost during the procedure. Place the mice under a heat lamp until their resuscitation.&#160;Note: Optimally, a safer warming device (such as a circulating warm water pad) can be used instead of a heat lamp.</li>
	<li>Postoperatively inject buprenorphin (0.05 mg/kg body weight s.c.) to achieve analgesia. Note: An animal should not be left unattended until it has regained sufficient consciousness to maintain sternal recumbency. An animal which has undergone a surgical treatment should not be returned to the company of other animals until fully recovered.</li>
	<li>Place animals back in their cages with unlimited access to food and water. Mice will be euthanized when they become moribund (indicated by clinical signs such as failure to move when touched or cyanosis). Check mice every 4 hr&#160;to avoid having them die prior to euthanasia.</li>
</ol>
2. Tissue Harvest and Fixation
<ol>
	<li>Prepare a 4% paraformaldehyde (PFA) solution: To 889 ml PBS add 111 ml of 37% PFA.</li>
	<li>Sacrifice mice using either CO2-induced narcosis or Ketamine/Xylazine solution in a high dosage.</li>
	<li>Immobilize the mice onto a clean work surface. Spray the abdominal and thoracic skin with 70% EtOH solution to disinfect it.</li>
	<li>Use a scalpel and small scissors to remove the abdominal and thoracic skin as well as the skin covering the tracheal area.</li>
	<li>Reveal trachea by removing its surrounding tissues (muscles and connective tissue).</li>
	<li>Place a suture (without cutting needle) around the trachea. Do not tighten it yet.</li>
	<li>Use a small peripheral venous catheter (20 G) to &#39;intubate&#39; trachea. Be careful during the catheter insertion to avoid tracheal tear. Note that the largest diameter of trachea is just below the cricoid cartilage.</li>
	<li>Tighten the suture around the trachea/catheter. Remove the needle leaving only the plastic cannula of the catheter into the trachea. Needle served only as a guide-wire for inserting the plastic cannula.</li>
	<li>After having secured the plastic cannula into the trachea, cut the abdominal musculature, open the abdomen and reveal the diaphragm.</li>
	<li>Using a needle, puncture the diaphragm to produce pneumothorax (i.e.&#160;insertion of air into the pleural cavity). Lungs should then be collapsed.</li>
	<li>Using small scissors, remove the diaphragm, cut along the sternum (sternotomy), remove the rib cage and reveal the heart and the lungs.</li>
	<li>Through the plastic cannula inserted into the trachea, fix the lungs with the formaldehyde solution under 30 cm H2O pressure.</li>
	<li>Remove the lungs and place them into the 4% PFA for 24 hr and subsequently in 70% EtOH solution until processing.</li>
	<li>Identify and remove heart, liver and kidneys. In the same way, place them into the 4% PFA for 24 hr and subsequently in 70% EtOH solution until processing.</li>
</ol>
3. GFP Mice and Viewing of GFP Slides
<ol>
	<li>Perform CLP and sham surgery on GFP-LC3 mice as described in step 1, and postoperative procedures as described in step 2 of the protocol.</li>
	<li>Embed the tissues (namely lungs, heart, liver and kidneys) in paraffin and cut them in serial 5&#160;&#956;m sections.</li>
	<li>When ready for GFP staining, incubate at 60 &#176;C for 30-60 min until paraffin melts and tissue appears translucent.</li>
	<li>Deparaffinize with standard graded series of xylene or xylene substitutes, 5 min xylene 1, xylene 2, and xylene 3 then 3 min incubations in graded series of EtOH: 100%, 95%, 75%, and then rinse in distilled water. 
	Note: After deparaffinization avoid tissue desiccation, which increases sample background.</li>
	<li>Perform antigen retrieval using citric acid buffer (10 mM sodium citrate acid, 0.05% Tween 20, pH 6.0), by microwaving for 10-20 min. Be careful to replace buffer periodically to avoid tissue desiccation.</li>
	<li>Allow solution to cool for 20 min on the bench to complete antigen retrieval.</li>
	<li>Wash 2-3x with PBS. Remove excess buffer and transfer slides from rack to horizontal position in a slide box or other humidified chamber to prevent desiccation.</li>
	<li>Block 45-60 min at room temperature with 10% normal donkey serum (also 5% bovine serum albumin, or preferably serum from the animal in which the secondary antibody was generated) in PBS.</li>
	<li>Incubate samples in primary GFP antibody at 1:100 dilution in PBS overnight at 4 &#176;C in humidified chamber. Gently apply small pieces of Parafilm over tissue with antibody to promote homogenous contact with the tissue and prevent evaporation. 
	Note: Remaining steps are performed at room temperature.</li>
	<li>Wash 5x with PBS to remove excess primary antibody. Incubate with secondary antibody 1:500-1:1,000 in PBS for 1-2 hr at room temperature.</li>
	<li>Wash 5x with PBS to remove excess secondary antibody. If desired, counterstain the slides at this step.</li>
	<li>If using fluorescent secondary antibody, incubate 0.1% Sudan Black in 70% EtOH for 15-20 min to reduce autofluorescence. Wipe off excess Sudan Black from around tissue and wash 5x with PBS over 20 min.</li>
	<li>Mount sample with coverslip and store horizontally, until mounting solution has set.</li>
</ol>
4. Alternative Protocol: Direct Detection of GFP-LC3
<ol>
	<li>Cannulate and inflate lungs with a ~500 ml of 50/50 v/v OCT PBS.</li>
	<li>Embed in OCT by putting a small amount of OCT in the bottom of a cryomold.</li>
	<li>Carefully place the dissected lung complex in the mold.</li>
	<li>Slowly freeze over methylbutane chilled using dry ice.</li>
	<li>Add more OCT until the tissue is completely covered and frozen solid. Keep on dry ice and store at -80 &#176;C.</li>
	<li>Using a cryomicrotome, cut 5-10 &#956;m thick sections protecting the sections from light. Store sections and remaining tissue at -80 &#176;C.</li>
	<li>To prepare slides for imaging, remove section from freezer and immediately apply a drop of PBS to prevent desiccation.</li>
	<li>Fix for 30 min with 4% PFA. Wash with PBS.</li>
	<li>Apply DAPI/Hoescht for 10 min. Wash 3x&#160;with PBS.</li>
	<li>Mount sample with coverslip and store horizontally, until mounting solution has set.</li>
	<li>Image using epifluorescence or confocal microscope.&#160;Store samples at 4 &#176;C for up to one month.</li>
</ol>
5. Preparation of Tissue for Electron Microscopy
<ol>
	<li>Cut tissues into approximately 1 mm cubes for fixing.</li>
	<li>Fix the tissue in 2% formaldehyde, 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.4.</li>
	<li>Wash with 0.1 M sodium cacodylate buffer for 1-2 hr. 
	Note: Fixed tissue can be stored in 0.1 M sodium cacodylate at 4 &#176;C.</li>
	<li>Post fix in 1% osmium tetroxide in 0.2 M sodium cacodylate buffer for 1 hr.</li>
	<li>Rinse in 0.2 M sodium cacodylate buffer for 10 min 3x.</li>
	<li>Dehydrate in 70% EtOH, 20 min 2x. Dehydrate in 90% EtOH, 10 min 2x. Dehydrate in 100% EtOH, 20 min 2x.</li>
	<li>Incubate with propylene oxide (epoxy propane) for 10 min 2x.</li>
	<li>Incubate with propylene oxide/epoxy resin mixture (50:50) for 1 hr. The epoxy resin mixture is: 24 g Agar 100 resin, 13 g dodecenylsuccinic anhydride, 13 g methylnadic anhydride and 1 ml N-benzyldimethylamine. Mix thoroughly in a disposable beaker using a wooden spatula.</li>
	<li>Incubate with Epoxy resin overnight in uncapped vials (this allows any remaining propylene oxide to evaporate).</li>
	<li>Embed in labeled capsules with freshly prepared resin. Polymerize at 60 &#176;C for 48 hr.</li>
	<li>Photograph tissue sections using a transmission electron microscope at 80 or 60 kV onto electron microscope film. Print the images onto photographic paper or store as digital media.</li>
	<li>Analyze autophagosome formation in intact cells containing nucleus. Low power fields (e.g. 2,000-4,000X) can be used to identify nucleated cells. Typically 6,800-10,000X images are used for this autophagosome quantification. Count autophagosomes per unit area from 15-30 fields for appropriate statistical representation. Autophagosomes have double membrane structure, where late stage autophogosomes resembled filled vacuoles.</li>
</ol>

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The authors have no competing financial interests to declare

The major advantage of CLP is that it allows researchers to investigate sepsis of different severities (i.e.&#160;from low- to mid- and high-grade). Severity of induced sepsis is affected by the length of cecum ligated (which the most important determinant), the size of needle used for puncture and the number of holes performed15. In addition, the mouse strain and gender can impact on the severity of sepsis; several strains are more susceptible than others and males are generally more susceptible than...

Sepsis, a systemic inflammatory response to infection, represents a leading cause of death in critically-ill patients1. Intra-abdominal infections, often leading to polymicrobial sepsis, account for 20% of sepsis cases, which have substantial mortality of up to 60%2. Sepsis-associated mortality primarily results from multi-organ dysfunction with subsequent organ failure3,4. Additional investigation into the pathogenic mechanism of this disease is urgently needed to promote the development of novel and more effective therapies.
The cecal ligation and puncture (CLP) method is a commonly used procedure for modeling sepsis in vivo. As the cecum is full of bacteria, its puncture results in polymicrobial peritonitis, translocation of bacteria into the blood (bacteremia), septic shock, multi-organ dysfunction and, ultimately, death5. It is generally accepted that CLP reflects clinical reality more accurately than previous techniques, such as injection of endotoxin or even purified bacteria into rodents, Thus, CLP is considered the gold standard (albeit not without limitations)6 for the experimental induction and, hence, the investigation of the pathogenesis of sepsis. In this monograph, we describe protocols designed to assess whether pathogenic mechanisms of sepsis include autophagy.
Autophagy, an evolutionarily conserved cellular process, facilitates the turnover of damaged proteins and organelles such as mitochondria and plays an important role in the clearance of intracellular pathogens including bacteria7,8. During autophagy, cytosolic proteins or organelles are sequestered into double membrane-bound vesicles called autophagosomes, which are subsequently delivered to the lysosomes for degradation9. A number of proteins have been identified as the mammalian homologues of autophagy-related genes (Atg), originally identified in yeast, which regulate the process of autophagy. The conversion of microtubule associated protein-1 light chain 3B (LC3B) (homologue of Atg8) from LC3B-I (free form) to LC3B-II (phosphatidylethanolamine-conjugated form) represents a major step in autophagosome formation9. Autophagic dysfunction is associated with aging and human diseases including cancer and neurodegenerative disorders10. Moreover, autophagy affects innate and adaptive immunity such as antigen presentation, lymphocyte development and cytokine secretion by immune cells8. Thus, it seems reasonable that autophagy might also play a role in the systemic inflammatory response to infection (i.e.&#160;in sepsis).
To date several methods have been described to assess the role of autophagy in tissue injury in vivo. These include the use of green fluorescence protein (GFP)-LC3 expressing mice&#160;and the quantification of autophagosomes in tissue by electron microscopy&#160;(these two methods are described in this monograph). Additional methods include the quantification of autophagic protein expression in tissue homogenates, and the analysis of autophagic flux (as described elsewhere)11-13. The goal of this review is to provide current protocols for assessing autophagy in vivo in the context of experimental sepsis.

<table border="0" cellpadding="0" cellspacing="0" >
	<tbody>
		<tr >
			<td >GFP-LC3 Transgenic Mice</td>
			<td >Riken (Japan)</td>
			<td >RBRC00806</td>
			<td >GFP-LC3#53</td>
		</tr>
		<tr >
			<td >Xylazine</td>
			<td >Henry-Schein</td>
			<td >568-0606</td>
			<td>Xylazine HCl Injection Vet</td>
		</tr>
		<tr >
			<td >Ketamine</td>
			<td >Henry-Schein</td>
			<td >995-2949</td>
			<td>Ketaset Inj 100 mg/ml</td>
		</tr>
		<tr >
			<td >EtOH</td>
			<td >Fisher</td>
			<td>A405-20</td>
			<td>Histology Grade</td>
		</tr>
		<tr >
			<td >EtOH</td>
			<td >Fisher</td>
			<td>A407-1</td>
			<td>For Sterilizatiion</td>
		</tr>
		<tr >
			<td >silk surgical sutures 6-0</td>
			<td >Owens &#38; Minor</td>
			<td >2300-0078OG, 017624</td>
			<td></td>
		</tr>
		<tr >
			<td >buprenorphine-HCl</td>
			<td >Henry-Schein</td>
			<td >614-5157</td>
			<td>Buprenex Ampules</td>
		</tr>
		<tr >
			<td >paraformaldehyde (37%) solution</td>
			<td >JT Baker</td>
			<td >S898-09</td>
			<td></td>
		</tr>
		<tr >
			<td >xylenes</td>
			<td >Fisher</td>
			<td>X3P-1GAL </td>
			<td></td>
		</tr>
		<tr >
			<td >anti-GFP monoclonal antibody</td>
			<td>Life Technologies</td>
			<td>G10362</td>
			<td></td>
		</tr>
		<tr >
			<td >Hoescht</td>
			<td >Sigma</td>
			<td>944403</td>
			<td></td>
		</tr>
		<tr >
			<td >DAPI</td>
			<td >Invitrogen</td>
			<td >D1306</td>
			<td></td>
		</tr>
		<tr >
			<td >OCT</td>
			<td >VWR scientific</td>
			<td >25608-930</td>
			<td></td>
		</tr>
		<tr >
			<td >Sudan Black</td>
			<td >Santa Cruz</td>
			<td >sc-203760</td>
			<td ></td>
		</tr>
		<tr >
			<td >EM grade Glutaraldehyde 2.5% in sodium cacodylate</td>
			<td >Electron microscopy Sciences</td>
			<td>15960</td>
			<td></td>
		</tr>
		<tr >
			<td >propylene oxide</td>
			<td >Sigma</td>
			<td>240397</td>
			<td></td>
		</tr>
		<tr >
			<td >Agar 100 resin</td>
			<td >Agar scientific</td>
			<td > R1045</td>
			<td></td>
		</tr>
		<tr >
			<td >dodecenylsuccinic anhydride</td>
			<td >Sigma</td>
			<td>46346</td>
			<td></td>
		</tr>
		<tr >
			<td >methylnadic anhydride</td>
			<td >Sigma</td>
			<td>45359</td>
			<td></td>
		</tr>
		<tr >
			<td >N-benzyldimethylamine</td>
			<td >Sigma</td>
			<td>185582</td>
			<td></td>
		</tr>
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cecal ligation and puncture-induced sepsis as a model to study autophagy in mice

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

Bacteremia is present in mice as early as 6 hr after CLP-induced sepsis14. Clinical signs of sepsis (including chills, tachypnoea and impaired motor activity) appear approximately 12 hr after the procedure. Mice subjected to CLP begin to die at around 18 hr after induction of peritonitis. The more severe is the sepsis the more increased is the lethality15. In detail, high-grade sepsis causes 100% mortality within 2-3 days, while mid-grade sepsis results in around 60% mortality at 7 days after CLP (b...

Watch this Scientific Journal Video about Cecal Ligation and Puncture-induced Sepsis as a Model To Study Autophagy in Mice at JoVE.com

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

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

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

cecal-ligation-puncture-induced-sepsis-as-model-to-study-autophagy

Research

JoVE Journal

Immunology and Infection

28.2K visualizações.  Brigham and Women's Hospital.  Sepse experimental pode ser induzida em camundongos usando a ligadura e punção do ceco método (CLP). Protocolos correntes para avaliar a autofagia in vivo no contexto de sepsia induzida por CLP são aqui apresentados: Um protocolo para medir autofagia utilizando ratinhos (GFP)-LC3, e um protocolo para medir a formação de autophagosome por microscopia electrónica. 

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

Colon Ascendens Stent Peritonitis (CASP) - a Standardized Model for Polymicrobial Abdominal Sepsis

Sepsis remains a persistent problem on intensive care units all over the world. Understanding the complex mechanisms of sepsis is the precondition for establishing new therapeutic approaches in this field. Therefore, animal models are required that are able to closely mimic the human disease and also sufficiently deal with scientific questions. The Colon Ascendens Stent Peritonitis (CASP) is a highly standardized model for polymicrobial abdominal sepsis in rodents. In this model, a small stent is surgically inserted into the ascending colon of mice or rats leading to a continuous leakage of intestinal bacteria into the peritoneal cavity. The procedure results in peritonitis, systemic bacteraemia, organ infection by gut bacteria, and systemic but also local release of several pro- and anti-inflammatory cytokines. The lethality of CASP can be controlled by the diameter of the inserted stent. A variant of this model, the so-called CASP with intervention (CASPI), raises opportunity to remove the septic focus by a second operation according to common procedures in clinical practice. CASP is an easily learnable and highly reproducible model that closely mimics the clinical course of abdominal sepsis. It leads way to study on questions in several scientific fields e.g. immunology, infectiology, or surgery.

Colon Ascendens Stent Peritonitis CASP - a Standardized Model for Polymicrobial Abdominal Sepsis

The Colon Ascendens Stent Peritonitis (CASP) is a highly standardized model for polymicrobial abdominal sepsis in rodents. This article describes the surgical procedure of CASP. The CASP model and its variants allow the systematic investigation of various problems concerning the subject of sepsis.

Sepsis remains a persistent problem on intensive care units all over the world. Understanding the complex mechanisms of sepsis is the precondition for ...

Dissecting Host-virus Interaction in Lytic Replication of a Model Herpesvirus

In response to viral infection, a host develops various defensive responses, such as activating innate immune signaling pathways that lead to antiviral cytokine production1,2. In order to colonize the host, viruses are obligate to evade host antiviral responses and manipulate signaling pathways. Unraveling the host-virus interaction will shed light on the development of novel therapeutic strategies against viral infection.
Murine &gamma;HV68 is closely related to human oncogenic Kaposi's sarcoma-associated herpesvirus and Epsten-Barr virus3,4. &gamma;HV68 infection in laboratory mice provides a tractable small animal model to examine the entire course of host responses and viral infection in vivo, which are not available for human herpesviruses. In this protocol, we present a panel of methods for phenotypic characterization and molecular dissection of host signaling components in &gamma;HV68 lytic replication both in vivo and ex vivo. The availability of genetically modified mouse strains permits the interrogation of the roles of host signaling pathways during &gamma;HV68 acute infection in vivo. Additionally, mouse embryonic fibroblasts (MEFs) isolated from these deficient mouse strains can be used to further dissect roles of these molecules during &gamma;HV68 lytic replication ex vivo.
Using virological and molecular biology assays, we can pinpoint the molecular mechanism of host-virus interactions and identify host and viral genes essential for viral lytic replication. Finally, a bacterial artificial chromosome (BAC) system facilitates the introduction of mutations into the viral factor(s) that specifically interrupt the host-virus interaction. Recombinant &gamma;HV68 carrying these mutations can be used to recapitulate the phenotypes of &gamma;HV68 lytic replication in MEFs deficient in key host signaling components. This protocol offers an excellent strategy to interrogate host-pathogen interaction at multiple levels of intervention in vivo and ex vivo.
Recently, we have discovered that &gamma;HV68 usurps an innate immune signaling pathway to promote viral lytic replication5. Specifically, &gamma;HV68 de novo infection activates the immune kinase IKK&beta; and activated IKK&beta; phosphorylates the master viral transcription factor, replication and transactivator (RTA), to promote viral transcriptional activation. In doing so, &gamma;HV68 efficiently couples its transcriptional activation to host innate immune activation, thereby facilitating viral transcription and lytic replication. This study provides an excellent example that can be applied to other viruses to interrogate host-virus interaction.

We describe a protocol to identify key roles of host signaling molecules in lytic replication of a model herpesvirus, gamma herpesvirus 68 (&gamma;HV68). Utilizing genetically modified mouse strains and embryonic fibroblasts for &gamma;HV68 lytic replication, the protocol permits both phenotypic characterization and molecular interrogation of virus-host interactions in viral lytic replication.

In response to viral infection, a host develops various defensive responses, such as activating innate immune signaling pathways that lead to antiviral ...

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 ...

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 ...

Use of Galleria mellonella as a Model Organism to Study Legionella pneumophila Infection

Legionella pneumophila, the causative agent of a severe pneumonia named Legionnaires&#39; disease, is an important human pathogen that infects and replicates within alveolar macrophages. Its virulence depends on the Dot/Icm type IV secretion system (T4SS), which is essential to establish a replication permissive vacuole known as the Legionella containing vacuole (LCV). L. pneumophila infection can be modeled in mice however most mouse strains are not permissive, leading to the search for novel infection models. We have recently shown that the larvae of the wax moth Galleria mellonella are suitable for investigation of L. pneumophila infection. G. mellonella is increasingly used as an infection model for human pathogens and a good correlation exists between virulence of several bacterial species in the insect and in mammalian models. A key component of the larvae&#39;s immune defenses are hemocytes, professional phagocytes, which take up and destroy invaders. L. pneumophila is able to infect, form a LCV and replicate within these cells. Here we demonstrate protocols for analyzing L. pneumophila virulence in the G. mellonella model, including how to grow infectious L. pneumophila, pretreat the larvae with inhibitors, infect the larvae and how to extract infected cells for quantification and immunofluorescence microscopy. We also describe how to quantify bacterial replication and fitness in competition assays. These approaches allow for the rapid screening of mutants to determine factors important in L. pneumophila virulence, describing a new tool to aid our understanding of this complex pathogen.

Use of Galleria mellonella as a Model Organism to Study Legionella pneumophila Infection

The larva of the wax moth Galleria mellonella was recently established as an in vivo model to study Legionella pneumophila infection. Here, we demonstrate fundamental techniques to characterize the pathogenesis of Legionella in the larvae, including inoculation, measurement of bacterial virulence and replication as well as extraction and analysis of infected hemocytes.

Legionella pneumophila, the causative agent of a severe pneumonia named Legionnaires&#39; disease, is an important human pathogen that infects and ...

Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions

Shigella flexneri is an intracellular pathogen that can escape from phagosomes to reach the cytosol, and polymerize the host actin cytoskeleton to promote its motility and dissemination. New work has shown that proteins involved in actin-based motility are also linked to autophagy, an intracellular degradation process crucial for cell autonomous immunity. Strikingly, host cells may prevent actin-based motility of S. flexneri by compartmentalizing bacteria inside &#8216;septin cages&#8217; and targeting them to autophagy. These observations indicate that a more complete understanding of septins, a family of filamentous GTP-binding proteins, will provide new insights into the process of autophagy. This report describes protocols to monitor autophagy-cytoskeleton interactions caused by S. flexneri in vitro using tissue culture cells and in vivo using zebrafish larvae. These protocols enable investigation of intracellular mechanisms that control bacterial dissemination at the molecular, cellular, and whole organism level.

Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions

To counteract pathogen dissemination, host cells reorganize their cytoskeleton to compartmentalize bacteria and induce autophagy. Using Shigella infection of tissue culture cells, host and pathogen determinants underlying this process are identified and characterized. Using zebrafish models of Shigella infection, the role of discovered molecules and mechanisms are investigated in vivo.

Shigella flexneri is an intracellular pathogen that can escape from phagosomes to reach the cytosol, and polymerize the host actin cytoskeleton to promote ...

Transplantation of Tail Skin to Study Allogeneic CD4 T Cell Responses in Mice

The study of T cell responses and their consequences during allo-antigen recognition requires a model that enables one to distinguish between donor and host T cells, to easily monitor the graft, and to adapt the system in order to answer different immunological questions. Medawar and colleagues established allogeneic tail-skin transplantation in mice in 1955. Since then, the skin transplantation model has been continuously modified and adapted to answer specific questions. The use of tail-skin renders this model easy to score for graft rejection, requires neither extensive preparation nor deep anesthesia, is applicable to animals of all genetic background, discourages ischemic necrosis, and permits chemical and biological intervention. 
In general, both CD4+ and CD8+ allogeneic T cells are responsible for the rejection of allografts since they recognize mismatched major histocompatibility antigens from different mouse strains. Several models have been described for activating allogeneic T cells in skin-transplanted mice. The identification of major histocompatibility complex (MHC) class I and II molecules in different mouse strains including C57BL/6 mice was an important step toward understanding and studying T cell-mediated alloresponses. In the tail-skin transplantation model described here, a three-point mutation (I-Abm12) in the antigen-presenting groove of the MHC-class II (I-Ab) molecule is sufficient to induce strong allogeneic CD4+ T cell activation in C57BL/6 mice. Skin grafts from I-Abm12 mice on C57BL/6 mice are rejected within 12-15 days, while syngeneic grafts are accepted for up to 100 days. The absence of T cells (CD3-/- and Rag2-/- mice) allows skin graft acceptance up to 100 days, which can be overcome by transferring 2 x 104 wild type or transgenic T cells. Adoptively transferred T cells proliferate and produce IFN-&#947; in I-Abm12-transplanted Rag2-/- mice.

Tail-skin transplantation is a powerful model for studying T cell-dependent rejection and tolerance induction during allogeneic immune responses in mice. The advantages of this protocol are minor invasive surgery, and ease of monitoring with no need to sacrifice the recipient mouse.

The study of T cell responses and their consequences during allo-antigen recognition requires a model that enables one to distinguish between donor and ...

A Controlled Mouse Model for Neonatal Polymicrobial Sepsis

Neonatal sepsis remains a global burden. A preclinical model to screen effective prophylactic or therapeutic interventions is needed. Neonatal mouse polymicrobial sepsis can be induced by injecting cecal slurry intraperitoneally into day of life 7 mice and monitoring them for the following week. Presented here are the detailed steps necessary for the implementation of this neonatal sepsis model. This includes making a homogeneous cecal slurry stock, diluting it to a weight- and litter-adjusted dose, an outline of the monitoring schedule, and a definition of observed health categories used to define humane endpoints. The generation of a homogeneous cecal slurry stock from pooled donors allows for the administration into many litters over time, reducing the variation between donors, and preventing the use of potentially toxic glycerol. The monitoring strategy used allows for the anticipation of survival outcome and the identification of mice that would later progress to death, allowing for an earlier identification of the humane endpoint. Two main behavioral features are used to define the health scores, namely, the ability of the neonatal mice to right themselves when placed on their back and their level of mobility. These criteria could potentially be applied to address humane endpoints in other studies of neonatal disease in mice, as long as a pilot study is performed to confirm accuracy. In conclusion, this approach provides a standardized method to model newborn sepsis in mice, while providing resources to assess animal welfare used to define early humane endpoints for challenged animals.

This protocol provides the necessary steps to establish and evaluate neonatal sepsis in 7-day-old mice.

Neonatal sepsis remains a global burden. A preclinical model to screen effective prophylactic or therapeutic interventions is needed. Neonatal mouse ...

Design of Cecal Ligation and Puncture and Intranasal Infection Dual Model of Sepsis-Induced Immunosuppression

Sepsis, a severe and complicated life-threatening infection, is characterized by an imbalance between pro- and anti-inflammatory responses in multiple organs. With the development of therapies, most patients survive the hyperinflammatory phase but progress to an immunosuppressive phase, which increases the emergence of secondary infections. Therefore, improved understanding of the pathogenesis underlying secondary hospital-acquired infections in the immunosuppressive phase during sepsis is of tremendous importance. Reported here is a model to test infectious outcomes by creating double-hit infections in mice. A standard surgical procedure is used to induce polymicrobial peritonitis by cecal ligation and puncture (CLP) and followed by intranasal infection of Staphylococcus aureus to simulate pneumonia occurring in immune suppression that is frequently seen in septic patients. This dual model can reflect the immunosuppressive state occurring in patients with protracted sepsis and susceptibility to secondary infection from nosocomial pneumonia. Hence, this model provides a simple experimental approach to investigate the pathophysiology of sepsis-induced secondary bacterial pneumonia, which may be used for discovering novel treatments for sepsis and its complications.

This protocol describes techniques to measure infectious outcomes underlying secondary hospital-acquired infections in the immunosuppressive condition, first by establishing cecal ligation/puncture mice then challenging them with intranasal infection to create a clinically relevant model of immunosuppression sepsis.

Sepsis, a severe and complicated life-threatening infection, is characterized by an imbalance between pro- and anti-inflammatory responses in multiple ...

Use of the Invertebrate Galleria mellonella as an Infection Model to Study the Mycobacterium tuberculosis Complex

Tuberculosis is the leading global cause of infectious disease mortality and roughly a quarter of the world&#8217;s population is believed to be infected with Mycobacterium tuberculosis. Despite decades of research, many of the mechanisms behind the success of M. tuberculosis as a pathogenic organism remain to be investigated, and the development of safer, more effective antimycobacterial drugs are urgently needed to tackle the rise and spread of drug resistant tuberculosis. However, the progression of tuberculosis research is bottlenecked by traditional mammalian infection models that are expensive, time consuming, and ethically challenging. Previously we established the larvae of the insect Galleria mellonella (greater wax moth) as a novel, reproducible, low cost, high-throughput and ethically acceptable infection model for members of the M. tuberculosis complex. Here we describe the maintenance, preparation, and infection of G. mellonella with bioluminescent Mycobacterium bovis BCG lux. Using this infection model, mycobacterial dose dependent virulence can be observed, and a rapid readout of in vivo mycobacterial burden using bioluminescence measurements is easily achievable and reproducible. Although limitations exist, such as the lack of a fully annotated genome for transcriptomic analysis, ontological analysis against genetically similar insects can be carried out. As a low cost, rapid, and ethically acceptable model for tuberculosis, G. mellonella can be used as a pre-screen to determine drug efficacy and toxicity, and to determine comparative mycobacterial virulence prior to the use of conventional mammalian models. The use of the G. mellonella-mycobacteria model will lead to a reduction in the substantial number of animals currently used in tuberculosis research.

Use of the Invertebrate Galleria mellonella as an Infection Model to Study the Mycobacterium tuberculosis Complex

Galleria mellonella was recently established as a reproducible, cheap, and ethically acceptable infection model for the Mycobacterium tuberculosis complex. Here we describe and demonstrate the steps taken to establish successful infection of G. mellonella with bioluminescent Mycobacterium bovis BCG lux.

Tuberculosis is the leading global cause of infectious disease mortality and roughly a quarter of the world&#8217;s population is believed to be infected ...