All NMR spectra used as illustrative examples are derived from a previously published study7 which was financially supported by Nestl&eacute; .

1. Colonization of germ-free animals and sample collection
 <ol>
 <li>Remove germ-free animals from isolators and house them in a conventional husbandry room in cages equipped with filter in front of the conventional animals which will serve as controls (Figure 1).</li>
 <li>Mix half of the litter (3 days old) taken from the control conventional cage with the litter of the germ-free animals. Always keep 1/3 of the dirty conventional litter each time it is necessary to renew it in order to maintain a level of bacteria (keep it at least for 3 days).</li>
 <li>Collect urine in a 1.5 mL microtube by handling the mouse over the tube and help micturition by gently massage the bowel. Snap-freeze immediately in liquid nitrogen. Store at least at -40&deg;C until NMR analysis. A minimum volume of 20 &mu;L is required for acquisition with a 5 mm NMR probe, but it is recommended to use 30 &mu;L to improve quality of metabolic profiling.</li>
 <li>Animals should be euthanized without use of any anesthetic in order to avoid confounding NMR resonances due to hepatic metabolism of anesthetic compounds (for example, use cervical dislocation followed by confirmation of death by exsanguination) </li>
 </ol>
 2. Recommendation for collection of liver biopsy
 <ol>
 <li>Do not use any product containing alcohol to avoid contamination. Wash tools using water only or saline solution. </li>
 <li>Do not perforate gall bladder. In case of bile leak, wash tissue immediately with water or saline solution.</li>
 <li>Collect liver biopsies (about 15-50 mg) from the left lobe as shown in Figure 2. For reproducible biopsies, collect consistently in the center of the left lobe avoiding the peripheral areas where tissue is thinner. </li>
 <li>Snap freeze biopsies in liquid nitrogen immediately and store them at -80&deg;C until NMR analysis.</li>
 </ol>
 3. 1H NMR acquisition of urine microvolume
 <ol>
 <li>Prepare 0.2M sodium phosphate buffer solution in D2O (99.8 &#37;), pH 7.4 containing 1 mM 3-(trimethylsilyl)propionic acid-d4 (TSP).</li>
 <li>Mix 30 &mu;L of urine with 30 &mu;L of sodium phosphate buffer.</li>
 <li>Transfer 50 &mu;L of mixed solution into 1.7 mm NMR capillary tube (Figure 3 (2)) using a 50 &mu;L glass syringe equipped with a metal needle (OD 0.5 mm). Be careful to avoid bubbles.</li>
 <li>Fit the capillary adaptor (Figure 3 (3)) on top of the capillary containing urine sample and place it into a 2.5 mm NMR microtube for 5 mm NMR probe (Figure 3 (1)). Use this combination of tubes as a regular 5 mm NMR tube for spectral acquisition.</li>
 <li>Use the extraction rod (Figure 3 (4)) to remove capillary from 2.5 mm NMR tube by screwing the capillary adapter gently to pull it out.</li>
 </ol>
 4. 1H HR MAS NMR of liver tissue biopsy: sample preparation
 <ol>
 <li>MAS rotor components and tools are described in Figure 4.</li>
 <li>Insert biopsy (about 15-50 mg) into zirconium rotor (Figure 4 (1)) and fill the rest of the volume with pure D2O for NMR lock. Be careful to not make any bubbles because this would alter the quality of subsequent shimming and quality of data acquisition.</li>
 <li>Insert 50 &mu;L Teflon spacer (Figure 4 (2)) using the cylindrical screw (Figure 4 (5)). Unscrew it and calibrate it using the depth gauge on the short side (Figure 4 (8)). At this step, it is important to pay a specific attention to the sample because part of it can leak through the spacer hole. If this is the case, then part of the biopsy is destroyed and sample weight is no longer reliable. It is thus necessary to start again from the beginning the preparation of the sample.</li>
 <li>Place the thead pin (Figure 4 (3)) and screw it gently with the screwdriver (Figure 4 (6)). Dry out any residual water with a piece of tissue.</li>
 <li>Place the cap (Figure 4 (4)) at the top of the rotor and insert it in the rotor packer (Figure 4 (6)). Press firmly until the cap is in place. There should not be any space left between the rotor and its cap.</li>
 <li>Mark half of the bottom of the rotor using a black marker pen to allow optical spin rate detection.</li>
 <li>Place the rotor inside the NMR spectrometer and start spinning at 5 kHz. Acquire 1H NMR spectrum using CPMG pulse sequence13 according to manufacturer's guidelines. </li>
 <li>Use &alpha; anomeric glucose resonance at 5.22 ppm (doublet) to calibrate NMR spectra.</li>
 <li>To unpack the rotor, proceed by removing the cap using the cap remover (Figure 4 (9)). Unscrew thead pin and remove Teflon spacer using the cylindrical screw. Wash thoroughly using water and detergent.</li>
 </ol>
 5. Representative Results
 Gut microbial activity can be monitored using urinary metabolic profiling. A large number of urinary microbial co-metabolites identifiable by 1H NMR have been described in the literature7,14-17. These microbial co-metabolites are particularly useful to monitor the colonization process as they provide a quick and noninvasive way to estimate when the newly established ecosystem is stable. Figure 5A clearly illustrates the appearance of gut microbial co-metabolites over the colonization process. This figure shows a urinary metabolic profile obtained by following the procedure described in Step 2 for an animal colonized 20 days using procedure described in Step 1. This animal did not excrete any indoxyl sulfate and very little amounts of phenylacetylglycine (PAG) and p-cresol sulfate at the germ-free state (day 0-blue). As colonization progresses, these 3 markers of protein metabolism by the gut microbiota increase considerably to reach an equilibrium at day 20 (red). This is particularly easy to monitor for a group of animals as illustrated on Figure 5B using the PAG resonance. This diagram was obtained by integrating the area under the resonances highlighted in gray in Figure 5A (&delta; 7.40-7.43), corresponding to a specific resonance (triplet) of PAG for a group of 7 animals.
 1H High Resolution Magic Angle Spinning (HR MAS) NMR spectroscopy is a non destructive technique that allows quick and reproducible acquisitions of metabolic profiles of any kind of biopsy18. In this protocol, we used this powerful technique to obtain a hepatic metabolic profile of 2 mice before (blue) and after (red) colonization (Figure 6). This figure illustrates well the information that can be derived from a MAS NMR-based metabolic profile. Numerous amino acids as well as metabolites derived from energetic metabolism such as glucose, glycogen, lactate, triglycerides, (D)-3-hydroxybutyrate and nicotinurate can be visualized. These profiles also contain information relevant to oxidative stress (i.e. ascorbic acid, glutathione), nucleotide metabolism (i.e. inosine, uridine) and methylamine metabolism (i.e. choline, Trimethylamine-N-oxide). In this example, it is very clear that the germ-free mouse displays almost no glycogen and very low amounts of glucose and triglycerides as was previously published7.
 
 <img src="/files/ftp_upload/3642/3642fig1.jpg" alt="Figure 1."/> Figure 1. Overview of the colonization protocol. Germ-free and conventional animals are housed in cages equipped with filters side by side and their litters are exchanged to allow progressive colonization from the conventional gut microbiota (1). Gut microbial activity is monitored using 1H NMR-based metabolic profiling (2-3). Hepatic metabolism is assessed by 1H HR MAS NMR-based metabolic profiling (4-5).
 
 <img src="/files/ftp_upload/3642/3642fig2.jpg" alt="Figure 2."/> Figure 2. Mouse liver anatomy. The liver is displayed such as the flat side of the organ faces the table. For reproducible biopsies, it is advised to always collect samples from the center of the left lobe as indicated by the dashed rectangle.
 
 <img src="/files/ftp_upload/3642/3642fig3.jpg" alt="Figure 3."/> Figure 3. 1.7 mm NMR capillary kit to work with microvolumes. Key: 1: 2.5 mm NMR microtube, 2: 1.7 mm NMR capillary tube, 3: Capillary adapter, 4: Extraction rod.
 
 <img src="/files/ftp_upload/3642/3642fig4.jpg" alt="Figure 4."/> Figure 4. MAS rotor equipment. Key: 1: MAS rotor, 2: 50 &mu;L Teflon spacer, 3: Thead pin, 4: cap, 5: cylindrical screw, 6: screwdriver, 7: rotor packer, 8: depth gauge.
 
 <img src="/files/ftp_upload/3642/3642fig5.jpg" alt="Figure 5."/> Figure 5. Evolution of urinary metabolic profiles during colonization. 
 
 <ol class="ualpha">
 <li>Zoom on the aromatic region of the spectra between 6.8-7.8 ppm where microbial co-metabolites can be visualized. 1H NMR spectra were derived from a single individual at day 0 (blue), 4 (green), 15 (orange) and 20 (red) post-colonization. The grey zone corresponds to the area that was integrated to make the diagram in B.
Key: 1-MeHistamine: 1-methylhistamine; Indoxyl-S: Indoxyl sulfate; His: Histidine; p-Cresol-S: p-Cresol sulfate; PAG: Phenylacetylglycine.
</li>
 <li>Average PAG concentration during colonization (n=7). Student&rsquo;s t-test was used to compare the difference in PAG concentration at various time-points: a: p&lt;0.05 compared to day 0; b: p&lt;0.01 compared to day 10.</li>
 </ol>
 
 
 <img src="/files/ftp_upload/3642/3642fig6.jpg" alt="Figure 6."/> Figure 6.Typical 600 MHz 1H HR MAS NMR spectra of liver biopsies derived from germ-free (blue) and ex-germ-free (red) mice. Bold protons are responsible for the triglyceride resonance. 
Key: 3-HB: 3-hydroxybutyrate, GSH: reduced glutathione, TGs: Triglycerides, TMAO: Trimethylamine-N-oxide.

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We have nothing to disclose.

In this protocol, we described a progressive colonization procedure in an open environment to further investigate the impact of gut microbiota on hepatic metabolism assessed by 1H HR MAS NMR profiling of intact biopsy. Various methods of colonization have been described in the literature. The most common methods to colonize animals with a defined microbiota are oral gavage or contaminated drinking water19,20. Fecal inoculation can also be used as previously described21. The colonization m...

Table of specific reagents and equipment:
		<table border="1">
			<tbody>
				<tr>
					<th>Name of the reagent</th>
					<th>Company</th>
					<th>Catalogue number</th>
					<th>Comments</th>
				</tr>
				<tr>
					<td>2.5 mm microtube</td>
					<td>New Era</td>
					<td>NE-H5/2.5-V-Br</td>
					<td></td>
				</tr>
				<tr>
					<td>1.7 mm capillary tube</td>
					<td>Sigma-Aldrich</td>
					<td>NORS175001</td>
					<td></td>
				</tr>
				<tr>
					<td>Capillary adapter</td>
					<td>New Era</td>
					<td>NE-325-5/1.7</td>
					<td></td>
				</tr>
				<tr>
					<td>Extraction rod</td>
					<td>New Era</td>
					<td>NE-341-5</td>
					<td></td>
				</tr>
				<tr>
					<td>HR-MAS rotor BL4 with 50 &mu;L spherical Teflon spacer kit</td>
					<td>Bruker</td>
					<td>HZ07213</td>
					<td></td>
				</tr>
				<tr>
					<td>Tool kit for 50 &mu;L inserts</td>
					<td>Bruker</td>
					<td>B2950</td>
					<td></td>
				</tr>
				<tr>
					<td>Advance III 600 MHz NMR</td>
					<td>Bruker</td>
					<td></td>
					<td></td>
				</tr>
				<tr>
					<td>1H HR MAS NMR solid probe</td>
					<td>Bruker</td>
					<td></td>
					<td></td>
				</tr>
				<tr>
					<td>Deuterium oxide 99.9 &#37;</td>
					<td>Sigma-Aldrich</td>
					<td>530867-1L</td>
					<td></td>
				</tr>
				<tr>
					<td>3-(trimethylsilyl)propionic acid-d4 (TSP)</td>
					<td>Sigma-Aldrich</td>
					<td>269913</td>
					<td></td>
				</tr>
			</tbody>
		</table>

assessing hepatic metabolic changes during progressive colonization of germ-free mouse by 1h nmr spectroscopy

It is well known that gut bacteria contribute significantly to the host homeostasis, providing a range of benefits such as immune protection and vitamin synthesis. They also supply the host with a considerable amount of nutrients, making this ecosystem an essential metabolic organ. In the context of increasing evidence of the link between the gut flora and the metabolic syndrome, understanding the metabolic interaction between the host and its gut microbiota is becoming an important challenge of modern biology.1-4
 Colonization (also referred to as normalization process) designates the establishment of micro-organisms in a former germ-free animal. While it is a natural process occurring at birth, it is also used in adult germ-free animals to control the gut floral ecosystem and further determine its impact on the host metabolism. A common procedure to control the colonization process is to use the gavage method with a single or a mixture of micro-organisms. This method results in a very quick colonization and presents the disadvantage of being extremely stressful5. It is therefore useful to minimize the stress and to obtain a slower colonization process to observe gradually the impact of bacterial establishment on the host metabolism.
 In this manuscript, we describe a procedure to assess the modification of hepatic metabolism during a gradual colonization process using a non-destructive metabolic profiling technique. We propose to monitor gut microbial colonization by assessing the gut microbial metabolic activity reflected by the urinary excretion of microbial co-metabolites by 1H NMR-based metabolic profiling. This allows an appreciation of the stability of gut microbial activity beyond the stable establishment of the gut microbial ecosystem usually assessed by monitoring fecal bacteria by DGGE (denaturing gradient gel electrophoresis).6 The colonization takes place in a conventional open environment and is initiated by a dirty litter soiled by conventional animals, which will serve as controls. Rodents being coprophagous animals, this ensures a homogenous colonization as previously described.7
 Hepatic metabolic profiling is measured directly from an intact liver biopsy using 1H High Resolution Magic Angle Spinning NMR spectroscopy. This semi-quantitative technique offers a quick way to assess, without damaging the cell structure, the major metabolites such as triglycerides, glucose and glycogen in order to further estimate the complex interaction between the colonization process and the hepatic metabolism7-10. This method can also be applied to any tissue biopsy11,12.

Watch this Scientific Journal Video about Assessing Hepatic Metabolic Changes During Progressive Colonization of Germ-free Mouse by 1H NMR Spectroscopy at JoVE.com

Assessing Hepatic Metabolic Changes During Progressive Colonization of Germ-free Mouse by 1H NMR Spectroscopy

A progressive colonization procedure is described to further assess its impact on the host hepatic metabolism. Colonization is monitored non invasively by evaluating the urinary excretion of microbial co-metabolites by NMR-based metabolic profiling while hepatic metabolism is assessed by High Resolution Magic Angle Spinning (HR MAS) NMR profiling of intact biopsy.

Assessing Hepatic Metabolic Changes During Progressive Colonization of Germ-free Mouse by 1H NMR Spectroscopy

It is well known that gut bacteria contribute significantly to the host homeostasis, providing a range of benefits such as immune protection and vitamin ...

assessing-hepatic-metabolic-changes-during-progressive-colonization

Research

JoVE Journal

Immunology and Infection

13.0K Views.  The University of Reading. A progressive colonization procedure is described to further assess its impact on the host hepatic metabolism. Colonization is monitored non invasively by evaluating the urinary excretion of microbial co-metabolites by NMR-based metabolic profiling while hepatic metabolism is assessed by High Resolution Magic Angle Spinning (HR MAS) NMR profiling of intact biopsy.

Video: Assessing Hepatic Metabolic Changes During Progressive Colonization of Germ-free Mouse by 1H NMR Spectroscopy

Quantification of Fungal Colonization, Sporogenesis, and Production of Mycotoxins Using Kernel Bioassays

The rotting of grains by seed-infecting fungi poses one of the greatest economic challenges to cereal production worldwide, not to mention serious risks to human and animal health. Among cereal production, maize is arguably the most affected crop, due to pathogen-induced losses in grain integrity and mycotoxin seed contamination. The two most prevalent and problematic mycotoxins for maize growers and food and feed processors are aflatoxin and fumonisin, produced by Aspergillus flavus and Fusarium verticillioides, respectively.
Recent studies in molecular plant-pathogen interactions have demonstrated promise in understanding specific mechanisms associated with plant responses to fungal infection and mycotoxin contamination1,2,3,4,5,6. Because many labs are using kernel assays to study plant-pathogen interactions, there is a need for a standardized method for quantifying different biological parameters, so results from different laboratories can be cross-interpreted. For a robust and reproducible means for quantitative analyses on seeds, we have developed in-lab kernel assays and subsequent methods to quantify fungal growth, biomass, and mycotoxin contamination. Four sterilized maize kernels are inoculated in glass vials with a fungal suspension (106) and incubated for a predetermined period. Sample vials are then selected for enumeration of conidia by hemocytometer, ergosterol-based biomass analysis by high performance liquid chromatography (HPLC), aflatoxin quantification using an AflaTest fluorometer method, and fumonisin quantification by HPLC.

The devastation of cereal crops by seed-infecting fungi has prompted numerous research efforts to better understand plant-pathogen interactions. To study seed-fungal interactions in a laboratory setting, we developed a robust method for the quantification of fungal reproduction, biomass, and mycotoxin contamination using kernel bioassays.

The rotting of grains by seed-infecting fungi poses one of the greatest economic challenges to cereal production worldwide, not to mention serious risks ...

Colonization of Euprymna scolopes Squid by Vibrio fischeri

Specific bacteria are found in association with animal tissue1-5. Such host-bacterial associations (symbioses) can be detrimental (pathogenic), have no fitness consequence (commensal), or be beneficial (mutualistic). While much attention has been given to pathogenic interactions, little is known about the processes that dictate the reproducible acquisition of beneficial/commensal bacteria from the environment. The light-organ mutualism between the marine Gram-negative bacterium V. fischeri and the Hawaiian bobtail squid, E. scolopes, represents a highly specific interaction in which one host (E. scolopes) establishes a symbiotic relationship with only one bacterial species (V. fischeri) throughout the course of its lifetime6,7. Bioluminescence produced by V. fischeri during this interaction provides an anti-predatory benefit to E. scolopes during nocturnal activities8,9, while the nutrient-rich host tissue provides V. fischeri with a protected niche10. During each host generation, this relationship is recapitulated, thus representing a predictable process that can be assessed in detail at various stages of symbiotic development. In the laboratory, the juvenile squid hatch aposymbiotically (uncolonized), and, if collected within the first 30-60 minutes and transferred to symbiont-free water, cannot be colonized except by the experimental inoculum6. This interaction thus provides a useful model system in which to assess the individual steps that lead to specific acquisition of a symbiotic microbe from the environment11,12.
Here we describe a method to assess the degree of colonization that occurs when newly hatched aposymbiotic E. scolopes are exposed to (artificial) seawater containing V. fischeri. This simple assay describes inoculation, natural infection, and recovery of the bacterial symbiont from the nascent light organ of E. scolopes. Care is taken to provide a consistent environment for the animals during symbiotic development, especially with regard to water quality and light cues. Methods to characterize the symbiotic population described include (1) measurement of bacterially-derived bioluminescence, and (2) direct colony counting of recovered symbionts.

Colonization of Euprymna scolopes Squid by Vibrio fischeri

The method outlines the procedure by which the Hawaiian bobtail squid, Euprymna scolopes and its bacterial symbiont, Vibrio fischeri, are raised separately and then introduced to allow for specific colonization of the squid light organ by the bacteria. Colonization detection by bacterially-derived luminescence and by direct colony counting are described.

Specific bacteria are found in association with animal tissue1-5. Such host-bacterial associations (symbioses) can be detrimental (pathogenic), have no ...

Detection of MicroRNAs in Microglia by Real-time PCR in Normal CNS and During Neuroinflammation

Microglia are cells of the myeloid lineage that reside in the central nervous system (CNS)1. These cells play an important role in pathologies of many diseases associated with neuroinflammation such as multiple sclerosis (MS)2. Microglia in a normal CNS express macrophage marker CD11b and exhibit a resting phenotype by expressing low levels of activation markers such as CD45. During pathological events in the CNS, microglia become activated as determined by upregulation of CD45 and other markers3. The factors that affect microglia phenotype and functions in the CNS are not well studied. MicroRNAs (miRNAs) are a growing family of conserved molecules (~22 nucleotides long) that are involved in many normal physiological processes such as cell growth and differentiation4 and pathologies such as inflammation5. MiRNAs downregulate the expression of certain target genes by binding complementary sequences of their mRNAs and play an important role in the activation of innate immune cells including macrophages6 and microglia7. In order to investigate miRNA-mediated pathways that define the microglial phenotype, biological function, and to distinguish microglia from other types of macrophages, it is important to quantitatively assess the expression of particular microRNAs in distinct subsets of CNS-resident microglia. Common methods for measuring the expression of miRNAs in the CNS include quantitative PCR from whole neuronal tissue and in situ hybridization. However, quantitative PCR from whole tissue homogenate does not allow the assessment of the expression of miRNA in microglia, which represent only 5-15% of the cells of neuronal tissue. Hybridization in situ allows the assessment of the expression of microRNA in specific cell types in the tissue sections, but this method is not entirely quantitative. In this report we describe a quantitative and sensitive method for the detection of miRNA by real-time PCR in microglia isolated from normal CNS or during neuroinflammation using experimental autoimmune encephalomyelitis (EAE), a mouse model for MS. The described method will be useful to measure the level of expression of microRNAs in microglia in normal CNS or during neuroinflammation associated with various pathologies including MS, stroke, traumatic injury, Alzheimer's disease and brain tumors.

Microglia are resident macrophages that provide the first line of defense and immune surveillance of the central nervous system. MicroRNAs are regulatory molecules that play an important role in many physiological processes including activation and differentiation of macrophages. In this article, we describe the method for measurement of microRNAs in microglia.

Microglia are cells of the myeloid lineage that reside in the central nervous system (CNS)1. These cells play an important role in pathologies of many ...

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing the mucosal surface of the nasopharynx, where it can reside, multiply and eventually overcome host defences to invade to other tissues of the host. Establishment of an infection in the normally lower respiratory tract results in pneumonia. Alternatively, the bacteria can disseminate into the bloodstream causing bacteraemia, which is associated with high mortality rates2, or else lead directly to the development of pneumococcal meningitis. Understanding the kinetics of, and immune responses to, nasopharyngeal colonization is an important aspect of S. pneumoniae infection models.
Our mouse model of intranasal colonization is adapted from human models3 and has been used by multiple research groups in the study of host-pathogen responses in the nasopharynx4-7. In the first part of the model, we use a clinical isolate of S. pneumoniae to establish a self-limiting bacterial colonization that is similar to carriage events in human adults. The procedure detailed herein involves preparation of a bacterial inoculum, followed by the establishment of a colonization event through delivery of the inoculum via an intranasal route of administration. Resident macrophages are the predominant cell type in the nasopharynx during the steady state. Typically, there are few lymphocytes present in uninfected mice8, however mucosal colonization will lead to low- to high-grade inflammation (depending on the virulence of the bacterial species and strain) that will result in an immune response and the subsequent recruitment of host immune cells. These cells can be isolated by a lavage of the tracheal contents through the nares, and correlated to the density of colonization bacteria to better understand the kinetics of the infection.

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Colonization of the murine nasopharynx with Streptococcus pneumoniae and the subsequent extraction of adherent or recruited cells is described. This technique involves flushing the nasopharynx and collection of the fluid through the nares and is adaptable for various readouts, including differential cell quantification and analysis of mRNA expression in situ.

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing ...

Assessing Anti-fungal Activity of Isolated Alveolar Macrophages by Confocal Microscopy

The lung is an interface where host cells are routinely exposed to microbes and microbial products. Alveolar macrophages are the first-line phagocytic cells that encounter inhaled fungi and other microbes. Macrophages and other immune cells recognize Aspergillus motifs by pathogen recognition receptors and initiate downstream inflammatory responses. The phagocyte NADPH oxidase generates reactive oxygen intermediates (ROIs) and is critical for host defense. Although NADPH oxidase is critical for neutrophil-mediated host defense1-3, the importance of NADPH oxidase in macrophages is not well defined. The goal of this study was to delineate the specific role of NADPH oxidase in macrophages in mediating host defense against A. fumigatus. We found that NADPH oxidase in alveolar macrophages controls the growth of phagocytosed A. fumigatus spores4. Here, we describe a method for assessing the ability of mouse alveolar macrophages (AMs) to control the growth of phagocytosed Aspergillus spores (conidia). Alveolar macrophages are stained in vivo and ten days later isolated from mice by bronchoalveolar lavage (BAL). Macrophages are plated onto glass coverslips, then seeded with green fluorescent protein (GFP)-expressing A. fumigatus spores. At specified times, cells are fixed and the number of intact macrophages with phagocytosed spores is assessed by confocal microscopy.

A method to evaluate the ability of isolated mouse alveolar macrophages to control the growth of phagocytosed Aspergillus spores by confocal microscopy.

The lung is an interface where host cells are routinely exposed to microbes and microbial products. Alveolar macrophages are the first-line phagocytic ...

Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is capable of causing serious invasive infections in immunocompromised patients, the elderly, and pregnant women. The most common manifestations of listeriosis in humans include meningitis, encephalitis, and fetal abortion. A significant but much less documented sequelae of invasive L. monocytogenes infection involves the heart. The death rate from cardiac illness can be up to 35% despite treatment, however very little is known regarding L. monocytogenes colonization of cardiac tissue and its resultant pathologies. In addition, it has recently become apparent that subpopulations of L. monocytogenes have an enhanced capacity to invade and grow within cardiac tissue. This protocol describes in detail in vitro and in vivo methods that can be used for assessing cardiotropism of L. monocytogenes isolates. Methods are presented for the infection of H9c2 rat cardiac myoblasts in tissue culture as well as for the determination of bacterial colonization of the hearts of infected mice. These methods are useful not only for identifying strains with the potential to colonize cardiac tissue in infected animals, but may also facilitate the identification of bacterial gene products that serve to enhance cardiac cell invasion and/or drive changes in heart pathology. These methods also provide for the direct comparison of cardiotropism between multiple L. monocytogenes strains.

Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes

Listeria monocytogenes causes fetal infections in pregnant women and meningitis in susceptible populations. Subpopulations of bacteria can colonize cardiac tissue, causing myocarditis in patients and laboratory animals. Here we present a protocol that describes how to assess L. monocytogenes cardiac cell invasion in vitro and cardiac colonization in infected animals.

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is capable of causing serious invasive infections in immunocompromised ...

Selective Harvesting of Marginating-hepatic Leukocytes

Marginating-hepatic (MH) leukocytes (leukocytes adhering to the sinusoids of the liver), were shown to exhibit unique composition and characteristics compared to leukocytes of other immune compartments. Specifically, evidence suggests a distinct pro- and anti-inflammatory profile of the MH-leukocyte population and higher cytotoxicity of liver-specific NK cells (namely, pit cells) compared to circulating or splenic immunocytes in both mice and rats. The method presented herein enables selective harvesting of MH leukocytes by forced perfusion of the liver in mice and rats. In contrast to other methods used to extract liver-leukocytes, including tissue grinding and biological degradation, this method exclusively yields leukocytes from the liver sinusoids, uncontaminated by cells from other liver compartments. In addition, the perfusion technique better preserves the integrity and the physiological milieu of MH leukocytes, sparing known physiological responses to tissue processing. As many circulating malignant cells and infected cells are detained while passing through the liver sinusoids, physically interacting with endothelial cells and resident leukocytes, the unique MH leukocyte population is strategically located to interact, identify, and react towards aberrant circulating cells. Thus, selective harvesting of MH-leukocytes and their study under various conditions may advance our understanding of the biological and clinical significance of MH leukocytes, specifically with respect to circulating aberrant cells and liver-related diseases and cancer metastases.

Marginating-hepatic leukocytes exhibit unique characteristics and distinct immunological functions compared to other leukocyte populations. Here we describe a method for selective harvesting of this specific hepatic cell population, through forced perfusion of the liver of rats or mice. Marginating-hepatic leukocytes seem critical in determining susceptibility to hepatic-related diseases and metastases.

Marginating-hepatic (MH) leukocytes (leukocytes adhering to the sinusoids of the liver), were shown to exhibit unique composition and characteristics ...

A Murine Model of Group B Streptococcus Vaginal Colonization

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of 10 - 30% of adults. In immune-compromised individuals, including neonates, pregnant women, and the elderly, GBS may switch to an invasive pathogen causing sepsis, arthritis, pneumonia, and meningitis. Because GBS is a leading bacterial pathogen of neonates, current prophylaxis is comprised of late gestation screening for GBS vaginal colonization and subsequent peripartum antibiotic treatment of GBS-positive mothers. Heavy GBS vaginal burden is a risk factor for both neonatal disease and colonization. Unfortunately, little is known about the host and bacterial factors that promote or permit GBS vaginal colonization. This protocol describes a technique for establishing persistent GBS vaginal colonization using a single &#946;-estradiol pre-treatment and daily sampling to determine bacterial load. It further details methods to administer additional therapies or reagents of interest and to collect vaginal lavage fluid and reproductive tract tissues. This mouse model will further the understanding of the GBS-host interaction within the vaginal environment, which will lead to potential therapeutic targets to control maternal vaginal colonization during pregnancy and to prevent transmission to the vulnerable newborn. It will also be of interest to increase our understanding of general bacterial-host interactions in the female vaginal tract.

A Murine Model of Group B Streptococcus Vaginal Colonization

The purpose of this protocol is to imitate human group B Streptococcus (GBS) vaginal colonization in a murine model. This method may be used to investigate host immune responses and bacterial factors contributing to GBS vaginal persistence, as well as to test therapeutic strategies.

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of ...

Molecular Diffusion in Plasma Membranes of Primary Lymphocytes Measured by Fluorescence Correlation Spectroscopy

Fluorescence correlation spectroscopy (FCS) is a powerful technique for studying the diffusion of molecules within biological membranes with high spatial and temporal resolution. FCS can quantify the molecular concentration and diffusion coefficient of fluorescently labeled molecules in the cell membrane. This technique has the ability to explore the molecular diffusion characteristics of molecules in the plasma membrane of immune cells in steady state (i.e., without processes affecting the result during the actual measurement time). FCS is suitable for studying the diffusion of proteins that are expressed at levels typical for most endogenous proteins. Here, a straightforward and robust method to determine the diffusion rate of cell membrane proteins on primary lymphocytes is demonstrated. An effective way to perform measurements on antibody-stained live cells and commonly occurring observations after acquisition are described. The recent advancements in the development of photo-stable fluorescent dyes can be utilized by conjugating the antibodies of interest to appropriate dyes that do not bleach extensively during the measurements. Additionally, this allows for the detection of slowly diffusing entities, which is a common feature of proteins expressed in cell membranes. The analysis procedure to extract molecular concentration and diffusion parameters from the generated autocorrelation curves is highlighted. In summary, a basic protocol for FCS measurements is provided; it can be followed by immunologists with an understanding of confocal microscopy but with no other previous experience of techniques for measuring dynamic parameters, such as molecular diffusion rates.

A method to measure protein diffusion in membranes of primary immune cells using fluorescence correlation spectroscopy (FCS) is described. In this paper, the use of antibodies for fluorescent labeling is illustrated.

Fluorescence correlation spectroscopy (FCS) is a powerful technique for studying the diffusion of molecules within biological membranes with high spatial ...

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

Enterohemorrhagic E. coli (EHEC) O157:H7, which is a foodborne pathogen that causesdiarrhea, hemorrhagic colitis (HS), and hemolytic uremic syndrome (HUS), colonize to the intestinal tract of humans. To study the detailed mechanism of EHEC colonization in vivo, it is essential to have animal models to monitor and quantify EHEC colonization. We demonstrate here a mouse-EHEC colonization model by transforming the bioluminescent expressing plasmid to EHEC to monitor and quantify EHEC colonization in living hosts. Animals inoculated with bioluminescence-labeled EHEC show intense bioluminescent signals in mice by detection with a non-invasive in vivo imaging system. After 1 and 2 days post infection, bioluminescent signals could still be detected in infected animals, which suggests that EHEC colonize in hosts for at least 2 days. We also demonstrate that these bioluminescent EHEC locate to mouse intestine, specifically in the cecum and colon, from ex vivo images. This mouse-EHEC colonization model may serve as a tool to advance the current knowledge of the EHEC colonization mechanism.

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

A detailed protocol of a mouse model for enterohemorrhagic E. coli (EHEC) colonization by using bioluminescence-labeled bacteria is presented. The detection of these bioluminescent bacteria by a non-invasive in vivo imaging system in live animals can advance our current understanding of EHEC colonization.

Enterohemorrhagic E. coli (EHEC) O157:H7, which is a foodborne pathogen that causesdiarrhea, hemorrhagic colitis (HS), and hemolytic uremic syndrome ...