We thank Lee Tuddenham (IBMC, Strasbourg) for amplifying and titrating MCMV-Luc and Thomas Baumert (INSERM U748, Strasbourg) for permission to use the animal facility of the Institute of Virology. Financial support from INSERM, Universit&eacute; de Strasbourg and Agence Nationale de la Recherche (ANR-08-MIEN-005-01) is acknowledged. The initial participation of Sonia Beroud and Laetitia Lelieur during their Master project is also acknowledged.

1. Preparation of Viral Suspension
<ol>
	<li>Obtain the Smith strain of MCMV expressing Luciferase (MCMV-Luc 15) from Ulrich Koszninowski's laboratory. In this recombinant, the Luciferase gene is inserted in the ie2 locus of the MCMV genome.</li>
	<li>To amplify MCMV-Luc, infect a murine bone marrow stromal cell line (M2-10B4, ATCC #CRL-1972) with MCMV at different multiplicity of infection (MOI, 0.001 to 1) 16. For this, add virus to cultured cells in serum free medium at 37 &deg;C. After one hour, remove the supernatant and replace it with DMEM supplemented with 10% fetal calf serum (FCS). Five days later, harvest the culture medium and store aliquots at -80 &deg;C until further use.</li>
	<li>Titration of the viral suspension by plaque assay.
	<ol>
		<li>Split 3T3 cells (ATCC #CRL-1658) in a 24 well plate at 40,000 cells/well in 1 ml of DMEM supplemented with 10% FCS containing penicillin (200 IU/ml) and steptomycin (200 mg/ml) and incubate 24 hr at 37 &deg;C.</li>
		<li>Prepare serial dilutions (10-1 to 10-8) of an aliquot of MCMV suspension in DMEM and infect target cells (3T3) with 200 &mu;l of dilution after removing cell culture medium. Incubate 1 hr at 37 &deg;C. Add 1 ml of Top Medium (DMEM 3% FCS, 2% (v/v) gentamycin, 200 IU/ml penicillin, 200 mg/ml streptomycin, 8 g/L Carboxymethyl cellulose) and incubate 5 days at 37 &deg;C.</li>
		<li>Fix the cells by adding 200 &mu;l 10% formaldehyde (dilution of formaldehyde with water should be done in a chemical hood to avoid toxic inhalations) to each well, and incubating 6 hr at room temperature. Remove culture medium + fixator by pipetting out and add 200 &mu;l cell staining solution (1% crystal violet (w/v) in 20% ethanol). Before use, dilute 1 part with 9 parts milli-Q water. Incubate 2 min at room temperature, wash the plate 3-4x by soaking it in water, let dry and count the number of plaques with a binocular.</li>
	</ol>
	</li>
	<li>Dilute MCMV-Luc in DMEM to obtain 50 PFUs in 50 &mu;l and leave on ice until further use. Using higher viral inoculums induces lethality before the neonates reach 2-3 weeks of age. In our experience, 500 PFUs induce lethality in every neonate 5 to 7 days after injection.</li>
</ol>
2. Injection of Neonates
<ol>
	<li>When neonates (4 to 20 hr-old mice) are available, manipulate the litter and the feces of the cage with gloves before carefully handling the pups to avoid contamination with "foreign" odors that would stress the mother and induce the rejection of the pups.</li>
	<li>Perform an intraperitoneal injection using an insulin syringe (29 G) as shown in Figure 1A. Hold the neonate with the dorsal skin. With ventral side up, introduce the needle under the foreleg and gently push it subcutaneously to attain the peritoneal cavity (a little under the umbilicus). We currently inject 1% Methylene blue diluted in DPBS and monitor survival to practice the technique before performing viral infections. Since Methylene blue is clearly visible during the injection process, we recommend performing this "training" injection first to ensure proper delivery of the viral suspension in the intraperitoneal cavity.</li>
	<li>Eliminate the tiny blood drops that may arise and place the neonate back into the cage.</li>
</ol>
3. In vivo Imaging
<ol>
	<li>On day 7, handle the neonates with the same cautions as previously mentioned. Inject intraperitoneally a mixture of anesthetics (4 mg/ml ketamine, 0.8 mg/ml xylazine) and luciferin (0.15 mg/g body weight) in 50 &mu;l in each animal. Average body weight of the animals is 1-2 g and the dose of anesthetics per animal is: ketamine 40 &mu;g, xylazine 8 &mu;g Wait 12-15 min until luciferin reaches its maximum of emission. This has to be determined beforehand and depends on the luciferin provider and the imaging system used.</li>
	<li>While the pups are anesthetized, tag them for future identification and harvest a small piece of tissue that can then be used for future genotyping if mutant animals are used.</li>
	<li>Place the pups in the imaging system and perform acquisition of the light emitted by the whole body. Seven days after infection, the virus has replicated into numerous copies in target organs such as liver, kidneys, lungs and salivary glands; therefore, the duration of exposure should be short. In our case, we perform the acquisition for 10 to 20 sec (Figure 1B). The platform in the acquisition chamber of the imaging device needs to be heated to avoid excessive cooling of the body temperature of anesthetized animals. In addition, it is helpful to obtain low resolution images of several animals within one round of acquisition and then, move up the platform closer to the camera lens in order to focus on one part of a particular animal or an isolated organ following dissection. The software must enable rapid changes of the parameters (exposure time, distance sample/lens, sensitivity), quantification of defined regions of interest and future export of snapshot images.</li>
	<li>To acquire light signal from the head only, cover the body of the neonates lying laterally with a thick, dark paper that will mask the photons emitted at the level of the organs where high viral replication occurs (liver, lungs, salivary glands) and perform acquisition for 5 to 10 min (Figure 1C). Perform acquisition of the opposed side of the animal in the same conditions.</li>
	<li>Reintroduce the pups into the cage and place a heating lamp on top to avoid excessive cooling of the anesthetized neonates. Regularly monitor post-anesthetic recovery. With the indicated dose, anesthesia currently lasts 30 min.</li>
	<li>Repeat the same procedure on days 9, 11 and 14. On days 11 and 14, change the dose of anesthetics (6 mg/ml ketamine, 1.2 mg/ml xylazine).</li>
</ol>

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I., Baryawno, N., Soderberg-Naucler, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Is+human+cytomegalovirus+a+target+in+cancer+therapy?.">Is human cytomegalovirus a target in cancer therapy?.</a> Oncotarget. 2, 1329-1338 (2011).</li><li>Morein, B., Abusugra, I., Blomqvist, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunity+in+neonates.">Immunity in neonates.</a> Vet. Immunol. Immunopathol. 87, 207-213 (2002).</li><li>Zaghouani, H., Hoeman, C. M., Adkins, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neonatal+immunity:+faulty+T-helpers+and+the+shortcomings+of+dendritic+cells.">Neonatal immunity: faulty T-helpers and the shortcomings of dendritic cells.</a> Trends Immunol. 30, 585-591 (2009).</li><li>Morein, B., Blomqvist, G., Hu, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+responsiveness+in+the+neonatal+period.">Immune responsiveness in the neonatal period.</a> J. Comp. Pathol. 137, 27-31 (2007).</li><li>Cheeran, M. C., Lokensgard, J. R., Schleiss, M. 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Genome. 17, 398-406 (2006).</li><li>Juanjuan, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+model+for+congenital+CMV+infection+and+hearing+impairment.">Murine model for congenital CMV infection and hearing impairment.</a> Virol. J. 8, 70 (2011).</li><li>Koontz, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+development+of+the+brain+after+focal+herpesvirus+infection+of+the+central+nervous+system.">Altered development of the brain after focal herpesvirus infection of the central nervous system.</a> J. Exp. Med. 205, 423-435 (2008).</li><li>Bantug, G. 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The authors declare no competing financial interest.	
	Ethics statement	
	Animals were maintained under pathogen-free conditions in the animal care facility of the Institut d'Immunologie et d'H&eacute;matologie. Handling of mice and experimental procedures were conducted in accordance with the French Law for the Protection of Laboratory Animals. The procedure was approved by the service v&eacute;terinaire de la Pr&eacute;fecture du Bas-Rhin (France) under the authorization number A-67-345.

Using in vivo imaging technology to monitor MCMV-Luc dissemination in mice neonates, we were able to observe viral spread to the brain of mutant animals, as opposed to wild-type. Further dissection of the animal and ex vivo imaging of the brain confirmed the presence of luminescent virus in the central nervous system. In addition, we also performed immunohistochemistry (not shown) on brain thin sections with an antibody specific for MCMV E1 early protein and observed that indeed, MCMV is present in the ...

Human Cytomegalovirus (HCMV/HHV-5) is a member of the &beta;-herpesvirus family. HCMV is a highly prevalent, opportunistic pathogen which is usually acquired during early life as an asymptomatic infection 1. Like all herpesviruses, HCMV persists throughout the entire life of the host whose immune system tightly controls viral replication. Episodes of viral reactivation mostly occur in immunocompromised individuals such as transplant patients receiving drugs to prevent graft rejection 2. In adults, HCMV has also been linked to glioblastomas 3. In addition, HCMV is a prominent pathogen for newborns with immature immunity 4-6. Primary infection in the developing fetus or neonate can have severe consequences. HCMV infection is the most common infectious cause of congenital birth defects and childhood disorders in developed countries. It is estimated that the incidence of neonatal HCMV infection affects 0.5-1% of all live births among which 5-10% will suffer from severe symptoms such as microcephaly or cerebellar hypoplasia. In addition, 10% of the infected infants with subclinical viral infection will later develop sequellae leading to mental retardation, hearing loss, visual defects or seizure and epilepsy 7,8.	
	As opposed to other human herpesviruses such as Herpes Simplex 1 (HSV-1/HHV-1) which can be inoculated to mice via different routes of injection 9, cytomegalovirus replication is species specific. This feature has severely hampered investigations of HCMV pathogenesis which are performed in different animal models (mouse, rat, guinea pig, rhesus monkey) and their respective genuine host-specific CMVs. All CMVs exhibit significant similarities in genome size and organization, tissue tropism and regulation of gene expression. They also induce similar pathologies in their respective host. Despite genomic diversity between HCMV and mouse cytomegalovirus (MCMV) (50% of the ORFs present in the human virus are identified in the murine CMV), the mouse model has recently proved to be advantageous, mostly because mutant strains can be tested for their ability to control viral replication in vivo. This has led to a genetic screen which enabled an estimation of the number of mouse genes expressed at the adult stage which compose the "resistome" to this virus 10. Altogether, this indicates that MCMV-infected mice represent an attractive model for the study of host-virus interactions in adults. The exploration of congenital CMV infection is more complex because differences in placental layer organization between human and mice impair the mother-to-fetus transmission of the viral infection in mice. Recently, direct injection of MCMV in the placenta on day 12.5 of gestation has enabled brain infection of mice neonates which led to hearing impairment 11. However, most investigations now use intraperitoneal injection of 4-20 hr-old neonates to provide systemic viral dissemination potentially leading to hematogenous brain infection, a model which is more relevant than that of an intracranial injection. This protocol provided important insights into CMV pathogenesis and more particularly, it was demonstrated that MCMV infection of newborns results in viral replication in neuronal and glial cells located in inflammatory foci which are infiltrated with mononuclear cells like macrophages 12. This report also described altered morphogenesis of the cerebellum accompanied with diminished granular neuron proliferation and migration and the induction of multiple interferon-stimulated genes. The essential role of CD8+T cells for the control of MCMV in the central nervous system was also reported by the same group 13. An important aspect to consider when analyzing the pathological effect of a microbe is the dynamics of the infection. In the case of MCMV, it is particularly crucial to explore and quantify the progression of viral dissemination into the developing brain in order to understand and anticipate the magnitude of the future neurobiological injuries. Traditionally, the quantification of the progression of an infection requires the regular sacrifice of infected animals to titer the pathogen in tissues, such as the brain, which are otherwise inaccessible. This type of protocol is now challenged by necessary improvement of animal welfare and the 3Rs (Reduce, Refine, Replace) principles 14. Using in vivo imaging technologies may allow a drastic reduction of the number of animals which are necessary in in vivo infection experiments. Here, we report and describe a time-course analysis of viral dissemination into the brain upon intraperitoneal MCMV-Luc injection to mouse neonates. Using the same animals, we tracked and monitored in vivo the sites of intense viral replication during a 2-week period.

<table border="1">
		<tbody>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Reagent/Material</td>
		 </tr>
		 <tr>
			<td>DMEM</td>
			<td>Fisher Scientific</td>
			<td>W3523A</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Methylene blue</td>
			<td>Sigma Aldrich</td>
			<td>319112</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Insulin needles</td>
			<td>VWR</td>
			<td>613-4897</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Ketamine</td>
			<td>CentraVet</td>
			<td>Ket 201</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Xylazine/Vetranal</td>
			<td>Sigma Aldrich</td>
			<td>46995</td>
			<td></td>
		 </tr>
		 <tr>
			<td>DPBS</td>
			<td>DUTSCHER</td>
			<td>P0436500</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Luciferin</td>
			<td>Caliper</td>
			<td>760504</td>
			<td></td>
		 </tr>
		 <tr>
			<td>gentamycin</td>
			<td>Sigma Aldrich</td>
			<td>G1272</td>
			<td></td>
		 </tr>
		 <tr>
			<td>penicillin/streptomycin</td>
			<td>Gibco</td>
			<td>15070</td>
			<td></td>
		 </tr>
		 <tr>
			<td>carboxymethylcellulose</td>
			<td>Sigma Aldrich</td>
			<td>C4888</td>
			<td></td>
		 </tr>
		 <tr>
			<td>formaldehyde</td>
			<td>Sigma Aldrich</td>
			<td>F8775</td>
			<td></td>
		 </tr>
		 <tr>
			<td>crystal violet</td>
			<td>Sigma Aldrich</td>
			<td>C3886</td>
			<td></td>
		 </tr>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Equipment</td>
		 </tr>
		 <tr>
			<td>IVIS 50</td>
			<td>Caliper/Perkin Elmer</td>
			<td></td>
			<td></td>
		 </tr>
		</tbody>
 </table>

use of in vivo imaging to monitor the progression of experimental mouse cytomegalovirus infection in neonates

Human Cytomegalovirus (HCMV or HHV-5) is a life-threatening pathogen in immune-compromised individuals. Upon congenital or neonatal infection, the virus can infect and replicate in the developing brain, which may induce severe neurological damage, including deafness and mental retardation. Despite the potential severity of the symptoms, the therapeutic options are limited by the unavailability of a vaccine and the absence of a specific antiviral therapy. Furthermore, a precise description of the molecular events occurring during infection of the central nervous system (CNS) is still lacking since observations mostly derive from the autopsy of infected children. Several animal models, such as rhesus macaque CMV, have been developed and provided important insights into CMV pathogenesis in the CNS. However, despite its evolutionary proximity with humans, this model was limited by the intracranial inoculation procedure used to infect the animals and consistently induce CNS infection. Furthermore, ethical considerations have promoted the development of alternative models, among which neonatal infection of newborn mice with mouse cytomegalovirus (MCMV) has recently led to significant advances. For instance, it was reported that intraperitoneal injection of MCMV to Balb/c neonates leads to infection of neurons and glial cells in specific areas of the brain. These findings suggested that experimental inoculation of mice might recapitulate the deficits induced by HCMV infection in children. Nevertheless, a dynamic analysis of MCMV infection of neonates is difficult to perform because classical methodology requires the sacrifice of a significant number of animals at different time points to analyze the viral burden and/or immune-related parameters. To circumvent this bottleneck and to enable future investigations of rare mutant animals, we applied in vivo imaging technology to perform a time-course analysis of the viral dissemination in the brain upon peripheral injection of a recombinant MCMV expressing luciferase to C57Bl/6 neonates.

A representative experiment is illustrated in Figure 1. Upon intraperitoneal injection of 50 PFUs of MCMV-Luc (Panel A shows a similar injection performed with Methylene blue to visualize the subcutaneous route of the needle), neonates were anesthetized and received simultaneously 0.3 mg of the luciferase substrate (Luciferin, Caliper). Fifteen minutes later, animals were placed ventral side up in the acquisition chamber of the IVIS 50 (In Vivo Imaging System, Caliper) and the light emitted by the whole ...

Watch this Scientific Journal Video about Use of In vivo Imaging to Monitor the Progression of Experimental Mouse Cytomegalovirus Infection in Neonates at JoVE.com

Use of In vivo Imaging to Monitor the Progression of Experimental Mouse Cytomegalovirus Infection in Neonates

Human Cytomegalovirus (HCMV) infection of neonates represents an important cause of mental retardation, yet the molecular events leading to virus-induced pathogenesis are still poorly understood. To investigate the dynamics of brain infection, we adapted whole-animal in vivo imaging to perform time-course analysis of neonates infected with a luciferase-recombinant virus.

Use of In vivo Imaging to Monitor the Progression of Experimental Mouse Cytomegalovirus Infection in Neonates

Human Cytomegalovirus (HCMV or HHV-5) is a life-threatening  pathogen in immune-compromised individuals. Upon congenital or neonatal  infection, the virus ...

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Research

JoVE Journal

Immunology and Infection

14.9K Views.  Université de Strasbourg. الفيروس المضخم للخلايا البشرية (HCMV) إصابة حديثي الولادة يمثل أحد أهم أسباب التخلف العقلي، إلا أن الأحداث الجزيئية التي تؤدي إلى فيروس من صنع المرضية لا تزال غير مفهومة تماما. للتحقيق في ديناميات الإصابة في الدماغ، ونحن تكييفها كامل الحيوان في الجسم الحي...

Video: Use of In vivo Imaging to Monitor the Progression of Experimental Mouse Cytomegalovirus Infection in Neonates

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

Distinct species of Leishmania, a protozoan parasite of the family Trypanosomatidae, typically cause different human disease manifestations. The most common forms of disease are visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL). Mouse models of leishmaniasis are widely used, but quantification of parasite burdens during murine disease requires mice to be euthanized at various times after infection. Parasite loads are then measured either by microscopy, limiting dilution assay, or qPCR amplification of parasite DNA. The in vivo imaging system (IVIS) has an integrated software package that allows the detection of a bioluminescent signal associated with cells in living organisms. Both to minimize animal usage and to follow infection longitudinally in individuals, in vivo models for imaging Leishmania spp. causing VL or CL were established. Parasites were engineered to express luciferase, and these were introduced into mice either intradermally or intravenously. Quantitative measurements of the luciferase driving bioluminescence of the transgenic Leishmania parasites within the mouse were made using IVIS. Individual mice can be imaged multiple times during longitudinal studies, allowing us to assess the inter-animal variation in the initial experimental parasite inocula, and to assess the multiplication of parasites in mouse tissues. Parasites are detected with high sensitivity in cutaneous locations. Although it is very likely that the signal (photons/second/parasite) is lower in deeper visceral organs than the skin, but quantitative comparisons of signals in superficial versus deep sites have not been done. It is possible that parasite numbers between body sites cannot be directly compared, although parasite loads in the same tissues can be compared between mice. Examples of one visceralizing species (L. infantum chagasi) and one species causing cutaneous leishmaniasis (L. mexicana) are shown. The IVIS procedure can be used for monitoring and analyzing small animal models of a wide variety of Leishmania species causing the different forms of human leishmaniasis.

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

An in vivo imaging system is used to generate quantitative measurements of murine infection with the Trypanosomatid protozoan Leishmania. This is a non-invasive and non-lethal method for detecting parasites expressing luciferase within many tissues throughout the course of chronic Leishmania spp. infection.

Distinct species of Leishmania, a protozoan parasite of the family Trypanosomatidae, typically cause different human disease manifestations. The most ...

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists. The value of 2P microscopy is that it provides rich spatiotemporal information regarding cell behaviors within intact tissues and in live mice. Leukocyte recruitment plays a significant role in host defense against infection and when unchecked, can contribute to inflammatory and autoimmune diseases. Studying leukocyte recruitment in vivo is technically challenging since cells are moving rapidly within vessels located deep within light scattering tissues. To date, most intravital imaging studies require surgical preparation to expose the blood vessels and tissues. To avoid the tissue damage and inflammation induced by surgery itself, here, we describe a non-invasive single-cell imaging approach that can be used to study leukocyte trafficking in the mouse footpad and phalanges. We discuss the technical aspects of our 2P imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Here, we describe a non-invasive two-photon (2P) microscopy approach to study leukocyte homing in the mouse footpad. We discuss the technical aspects of our tissue imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists.  The value of 2P microscopy is that it ...

The Use of Carboxyfluorescein Diacetate Succinimidyl Ester (CFSE) to Monitor Lymphocyte Proliferation

Carboxyfluorescein succinimidyl ester (CFSE) is an effective and popular means to monitor lymphocyte division1-3. CFSE covalently labels long-lived intracellular molecules with the fluorescent dye, carboxyfluorescein. Thus, when a CFSE-labeled cell divides, its progeny are endowed with half the number of carboxyfluorescein-tagged molecules and thus each cell division can be assessed by measuring the corresponding decrease in cell fluorescence via Flow cytometry. The capacity of CFSE to label lymphocyte populations with a high fluorescent intensity of exceptionally low variance, coupled with its low cell toxicity, make it an ideal dye to measure cell division. Since it is a fluorescein-based dye it is also compatible with a broad range of other fluorochromes making it applicable to multi-color flow cytometry. This article describes the procedures typically used for labeling mouse lymphocytes for the purpose of monitoring up to 8 cell divisions. These labeled cells can be used both for in vitro and in vivo studies.

The Use of Carboxyfluorescein Diacetate Succinimidyl Ester CFSE to Monitor Lymphocyte Proliferation

CFSE covalently labels long-lived intracellular molecules with the fluorescent dye, carboxyfluorescein. As such, when a CFSE-labeled cell divides, its progeny have half the amount of fluorescence, which can thereby be used to assess cell division. This article describes the procedures typically used for labeling mouse lymphocytes with CFSE.

Carboxyfluorescein succinimidyl ester (CFSE) is an effective and popular means to monitor lymphocyte division1-3.  CFSE covalently labels long-lived ...

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

There is growing concern about the relevance of in vitro antimicrobial susceptibility tests when applied to isolates of P. aeruginosa from cystic fibrosis (CF) patients. Existing methods rely on single or a few isolates grown aerobically and planktonically. Predetermined cut-offs are used to define whether the bacteria are sensitive or resistant to any given antibiotic1. However, during chronic lung infections in CF, P. aeruginosa populations exist in biofilms and there is evidence that the environment is largely microaerophilic2. The stark difference in conditions between bacteria in the lung and those during diagnostic testing has called into question the reliability and even relevance of these tests3.
 Artificial sputum medium (ASM) is a culture medium containing the components of CF patient sputum, including amino acids, mucin and free DNA. P. aeruginosa growth in ASM mimics growth during CF infections, with the formation of self-aggregating biofilm structures and population divergence4,5,6. The aim of this study was to develop a microtitre-plate assay to study antimicrobial susceptibility of P. aeruginosa based on growth in ASM, which is applicable to both microaerophilic and aerobic conditions.
 An ASM assay was developed in a microtitre plate format. P. aeruginosa biofilms were allowed to develop for 3 days prior to incubation with antimicrobial agents at different concentrations for 24 hours. After biofilm disruption, cell viability was measured by staining with resazurin. This assay was used to ascertain the sessile cell minimum inhibitory concentration (SMIC) of tobramycin for 15 different P. aeruginosa isolates under aerobic and microaerophilic conditions and SMIC values were compared to those obtained with standard broth growth. Whilst there was some evidence for increased MIC values for isolates grown in ASM when compared to their planktonic counterparts, the biggest differences were found with bacteria tested in microaerophilic conditions, which showed a much increased resistance up to a &gt;128 fold, towards tobramycin in the ASM system when compared to assays carried out in aerobic conditions.
 The lack of association between current susceptibility testing methods and clinical outcome has questioned the validity of current methods3. Several in vitro models have been used previously to study P. aeruginosa biofilms7, 8. However, these methods rely on surface attached biofilms, whereas the ASM biofilms resemble those observed in the CF lung9 . In addition, reduced oxygen concentration in the mucus has been shown to alter the behavior of P. aeruginosa2 and affect antibiotic susceptibility10. Therefore using ASM under microaerophilic conditions may provide a more realistic environment in which to study antimicrobial susceptibility.

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

Current diagnostic antimicrobial susceptibility testing relies on the planktonic growth of isolates in nutrient rich, aerobic conditions. Here, we employ an alternative artificial sputum medium to study antimicrobial susceptibility of Pseudomonas aeruginosa biofilms under both aerobic and microaerophilic conditions more representative of the cystic fibrosis lung.

There  is growing concern about the relevance of in vitro antimicrobial  susceptibility tests when applied to isolates of P. aeruginosa from  cystic ...

Two Methods of Heterokaryon Formation to Discover HCV Restriction Factors

Hepatitis C virus (HCV) is a hepatotropic virus with a host-range restricted to humans and chimpanzees. Although HCV RNA replication has been observed in human non-hepatic and murine cell lines, the efficiency was very low and required long-term selection procedures using HCV replicon constructs expressing dominant antibiotic-selectable markers1-5. HCV in vitro research is therefore limited to human hepatoma cell lines permissive for virus entry and completion of the viral life cycle. Due to HCVs narrow species tropism, there is no immunocompetent small animal model available that sustains the complete HCV replication cycle 6-8. Inefficient replication of HCV in non-human cells e.g. of mouse origin is likely due to lack of genetic incompatibility of essential host dependency factors and/or expression of restriction factors.
We investigated whether HCV propagation is suppressed by dominant restriction factors in either human cell lines derived from non-hepatic tissues or in mouse liver cell lines. To this end, we developed two independent conditional trans-complementation methods relying on somatic cell fusion. In both cases, completion of the viral replication cycle is only possible in the heterokaryons. Consequently, successful trans-complementation, which is determined by measuring de novo production of infectious viral progeny, indicates absence of dominant restrictions.
Specifically, subgenomic HCV replicons carrying a luciferase transgene were transfected into highly permissive human hepatoma cells (Huh-7.5 cells). Subsequently, these cells were co-cultured and fused to various human and murine cells expressing HCV structural proteins core, envelope 1 and 2 (E1, E2) and accessory proteins p7 and NS2. Provided that cell fusion was initiated by treatment with polyethylene-glycol (PEG), the culture released infectious viral particles which infected na&iuml;ve cells in a receptor-dependent fashion.
To assess the influence of dominant restrictions on the complete viral life cycle including cell entry, RNA translation, replication and virus assembly, we took advantage of a human liver cell line (Huh-7 Lunet N cells 9) which lacks endogenous expression of CD81, an essential entry factor of HCV. In the absence of ectopically expressed CD81, these cells are essentially refractory to HCV infection 10 . Importantly, when co-cultured and fused with cells that express human CD81 but lack at least another crucial cell entry factor (i.e. SR-BI, CLDN1, OCLN), only the resulting heterokaryons display the complete set of HCV entry factors requisite for infection. Therefore, to analyze if dominant restriction factors suppress completion of the HCV replication cycle, we fused Lunet N cells with various cells from human and mouse origin which fulfill the above mentioned criteria. When co-cultured cells were transfected with a highly fusogenic viral envelope protein mutant of the prototype foamy virus (PFV11) and subsequently challenged with infectious HCV particles (HCVcc), de novo production of infectious virus was observed. This indicates that HCV successfully completed its replication cycle in heterokaryons thus ruling out expression of dominant restriction factors in these cell lines. These novel conditional trans-complementation methods will be useful to screen a large panel of cell lines and primary cells for expression of HCV-specific dominant restriction factors.

We describe two methods for conditional trans-complementation of hepatitis C virus (HCV) assembly and the completion of the full viral life cycle, which rely on heterokaryon formation. These techniques are suitable to screen for cell lines that express dominant restriction factors, which preclude production of infectious HCV progeny.

Hepatitis C virus (HCV) is a hepatotropic virus with a host-range restricted to humans and chimpanzees. Although HCV RNA replication has been observed in ...

In Vivo Imaging Systems (IVIS) Detection of a Neuro-Invasive Encephalitic Virus

Modern advancements in imaging technology encourage further development and refinement in the way viral research is accomplished. Initially proposed by Russel and Burch in Hume's 3Rs (replacement, reduction, refinement), the utilization of animal models in scientific research is under constant pressure to identify new methodologies to reduce animal usage while improving scientific accuracy and speed. A major challenge to Hume's principals however, is how to ensure the studies are statistically accurate while reducing animal disease morbidity and overall numbers. Vaccine efficacy studies currently require a large number of animals in order to be considered statistically significant and often result in high morbidity and mortality endpoints for identification of immune protection. We utilized in vivo imaging systems (IVIS) in conjunction with a firefly bioluminescent enzyme to progressively track the invasion of the central nervous system (CNS) by an encephalitic virus in a murine model. Typically, the disease progresses relatively slowly, however virus replication is rapid, especially within the CNS, and can lead to an often, lethal outcome. Following intranasal infection of the mice with TC83-Luc, an attenuated Venezuelan equine encephalitis virus strain modified to expresses a luciferase gene; we are able to visualize virus replication within the brain at least three days before the development of clinical disease symptoms. Utilizing CNS invasion as a key encephalitic disease development endpoint we are able to quickly identify therapeutic and vaccine protection against TC83-Luc infection before clinical symptoms develop. With IVIS technology we are able to demonstrate the rapid and accurate testing of drug therapeutics and vaccines while reducing animal numbers and morbidity.

In Vivo Imaging Systems IVIS Detection of a Neuro-Invasive Encephalitic Virus

Utilizing luciferase and in vivo imaging systems (IVIS) as a novel means to identify disease endpoints before clinical developments occur. IVIS has allowed us to visualize in real time the invasion of encephalitic viruses over multiple days, providing a more accurate disease model for future study. It has also allowed us to identify the potential protective features of antivirals and vaccines faster than currently utilized animal models. The capability to utilize individual animals over multiple time points ensures reduced animal requirements, costs, and overall morbidity to the animals utilized ensuring a more humane and more scientific means of disease study.

Modern advancements in imaging technology encourage further development and refinement in the way viral research is accomplished. Initially proposed by ...

The Use of Fluorescent Target Arrays for Assessment of T Cell Responses In vivo

The ability to monitor T cell responses in vivo is important for the development of our understanding of the immune response and the design of immunotherapies. Here we describe the use of fluorescent target array (FTA) technology, which utilizes vital dyes such as carboxyfluorescein succinimidyl ester (CFSE), violet laser excitable dyes (CellTrace Violet: CTV) and red laser excitable dyes (Cell Proliferation Dye eFluor 670: CPD) to combinatorially label mouse lymphocytes into &#62;250 discernable fluorescent cell clusters. Cell clusters within these FTAs can be pulsed with major histocompatibility (MHC) class-I and MHC class-II binding peptides and thereby act as target cells for CD8+ and CD4+ T cells, respectively. These FTA cells remain viable and fully functional, and can therefore be administered into mice to allow assessment of CD8+ T cell-mediated killing of FTA target cells and CD4+ T cell-meditated help of FTA B cell target cells in real time in vivo by flow cytometry. Since &#62;250 target cells can be assessed at once, the technique allows the monitoring of T cell responses against several antigen epitopes at several concentrations and in multiple replicates. As such, the technique can measure T cell responses at both a quantitative (e.g.&#160;the cumulative magnitude of the response) and a qualitative (e.g.&#160;functional avidity and epitope-cross reactivity of the response) level. Herein, we describe how these FTAs are constructed and give an example of how they can be applied to assess T cell responses induced by a recombinant pox virus vaccine.

The Use of Fluorescent Target Arrays for Assessment of T Cell Responses In vivo

The ability to monitor T cell responses in detail in vivo is important for the development of our understanding of the immune response. Here we describe the use of fluorescent target arrays (FTAs) in an in vivo T cell assay that assesses &#62;250 parameters simultaneously by flow cytometry.

The ability to monitor T cell responses in vivo is important for the development of our understanding of the immune response and the design of ...

Imaging of In Situ Interferon Gamma Production in the Mouse Spleen following Listeria monocytogenes Infection

Cytokines are small proteins secreted by cells, mediating cell-cell communications that are crucial for effective immune responses. One characteristic of cytokines is their pleiotropism, as they are produced by and can affect a multitude of cell types. As such, it is important to understand not only which cells are producing cytokines, but also in which environment they do so, in order to define more specific therapeutics. Here, we describe a method to visualize cytokine production in situ following bacterial infection. This technique relies on imaging cytokine-producing cells in their native environment by confocal microscopy. To do so, tissue sections are stained for markers of multiple cell types together with a cytokine stain. Key to this method, cytokine secretion is blocked directly in vivo before harvesting the tissue of interest, allowing for detection of the cytokine that accumulated inside the producing cells. The advantages of this method are multiple. First, the microenvironment in which cytokines are produced is preserved, which could ultimately inform on the signals required for cytokine production and the cells affected by those cytokines. In addition, this method gives an indication of the location of the cytokine production in vivo, as it does not rely on artificial in vitro re-stimulation of the producing cells. However, it is not possible to simultaneously analyze cytokine downstream signaling in cells that receive the cytokine. Similarly, the cytokine signals observed correspond&#160;only to the time-window during which cytokine secretion was blocked. While we describe the visualization of the cytokine Interferon (IFN) gamma in the spleen following mouse infection by the intracellular bacteria Listeria monocytogenes, this method could potentially be adapted to the visualization of any cytokine in most organs.

Imaging of In Situ Interferon Gamma Production in the Mouse Spleen following Listeria monocytogenes Infection

Here, we describe a simple confocal imaging method to visualize the in situ localization of cells secreting the cytokine Interferon gamma in murine secondary lymphoid organs. This protocol can be extended for the visualization of other cytokines in diverse tissues.

Cytokines are small proteins secreted by cells, mediating cell-cell communications that are crucial for effective immune responses. One characteristic of ...

An Ex vivo Assay to Study Candida albicans Hyphal Morphogenesis in the Gastrointestinal Tract

Candida albicans hyphal morphogenesis in the gastrointestinal (GI) tract is tightly controlled by various environmental signals, and plays an important role in the dissemination and pathogenesis of this opportunistic fungal pathogen. However, methods to visualize fungal hyphae in the GI tract in vivo are challenging which limits the understanding of environmental signals in controlling this morphogenesis process. The protocol described here demonstrates a novel ex vivo method for visualization of hyphal morphogenesis in gut homogenate extracts. Using an ex vivo assay, this study demonstrates that cecal contents from antibiotic treated mice, but not from untreated control mice, promote C. albicans hyphal morphogenesis in the gut content. Further, adding back specific groups of gut metabolites to the cecal contents from antibiotic-treated mice differentially regulates hyphal morphogenesis ex vivo. Taken together, this protocol represents a novel method to identify and investigate the environmental signals that control C. albicans hyphal morphogenesis in the GI tract.

An Ex vivo Assay to Study Candida albicans Hyphal Morphogenesis in the Gastrointestinal Tract

The ex vivo assay described in this study using gut homogenate extracts and immunofluorescence staining represents a novel method to examine the hyphal morphogenesis of Candida albicans in the GI tract. This method can be utilized to investigate the environmental signals regulating morphogenetic transition in the gut.

Candida albicans hyphal morphogenesis in the gastrointestinal (GI) tract is tightly controlled by various environmental signals, and plays an important ...

Recurrent Escherichia coli Urinary Tract Infection Triggered by Gardnerella vaginalis Bladder Exposure in Mice

Recurrent urinary tract infections (rUTI) caused by uropathogenic Escherichia coli (UPEC) are common and costly. Previous articles describing models of UTI in male and female mice have illustrated the procedures for bacterial inoculation and enumeration in urine and tissues. During an initial bladder infection in C57BL/6 mice, UPEC establish latent reservoirs inside bladder epithelial cells that persist following clearance of UPEC bacteriuria. This model builds on these studies to examine rUTI caused by the emergence of UPEC from within latent bladder reservoirs. The urogenital bacterium Gardnerella vaginalis is used as the trigger of rUTI in this model because it is frequently present in the urogenital tracts of women, especially in the context of vaginal dysbiosis that has been associated with UTI. In addition, a method for in situ bladder fixation followed by scanning electron microscopy (SEM) analysis of bladder tissue is also described, with potential application to other studies involving the bladder.

Recurrent Escherichia coli Urinary Tract Infection Triggered by Gardnerella vaginalis Bladder Exposure in Mice

A mouse model of uropathogenic E. coli (UPEC) transurethral inoculation to establish latent intracellular bladder reservoirs and subsequent bladder exposure to G. vaginalis to induce recurrent UPEC UTI is demonstrated. Also demonstrated are the enumeration of bacteria, urine cytology, and in situ bladder fixation and processing for scanning electron microscopy.

Recurrent urinary tract infections (rUTI) caused by uropathogenic Escherichia coli (UPEC) are common and costly. Previous articles describing models of ...

Use of In vivo Imaging to Monitor the Progression of Experimental Mouse Cytomegalovirus Infection in Neonates

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