The authors are grateful to the communities in Yunnan province, People's Republic of China and Agboville, C&ocirc;te d'Ivoire, for their participation in the studies described above. We are indebted to the local field teams that have assisted in the testing and execution of the protocols. We are particularly grateful to the following colleagues from the Swiss Tropical and Public Health Institute: Yvette Endriss for providing the microscopic images, Hanspeter Marti and his team for laboratory support and Stefanie Krauth for her assistance during the video filming. Finally, we thank the two anonymous referees for a series of useful comments.

1. Kato-Katz Technique
<ol>
 <li>Place a standard Kato-Katz template with a hole for holding 41.7 milligrams (mg) of stool on a microscope slide.</li>
 <li>Scoop 2-3 grams (g) of a fresh fecal sample onto a piece of newspaper or aluminum foil, and press a piece of wire or plastic mesh on top to sieve it.</li>
 <li>Using a small plastic spatula, scrape the sieved material off the mesh and completely fill the hole in the Kato-Katz template with it. To remove excess fecal material, level the content of the hole with the spatula.</li>
 <li>Vertically remove the template without disturbing the fecal material now adhering to the microscope slide. The template and spatula can be cleaned in water with detergent, and reused.</li>
 <li>Place a piece of cellophane, pre-soaked in glycerine-malachite green solution for at least 24 hours (hr), over the fecal sample on the microscope slide.</li>
 <li>To spread the fecal material into a thick smear, gently press a clean microscope slide against the sample slide, evenly distributing the material within a circle of a diameter slightly smaller than the breadth of the microscope slide.</li>
 <li>Allow the slide to clear for 30-60 minutes (min), during which the slides must be kept away from direct sunlight. Then, systematically examine the thick smear under a light microscope (40-100x magnification). Count the number of STH eggs and stratify them by species (i.e., A.lumbricoides, T.trichiura, and hookworm). Of note, other helminth eggs (e.g. Schistosoma mansoni) can also be detected by this method and should be counted and recorded separately.</li>
 <li>To get a standardized estimate of the intensity of infection, conventionally expressed as number of eggs per gram of stool (EPG), multiply the egg count by 24 (24 x 41.7 mg &asymp; 1 g).</li>
</ol>
2. Baermann Technique
<ol>
 <li>Set up a stand that can hold a glass funnel and attach a rubber tube, closed with a clip, to the bottom of the funnel.</li>
 <li>Place a sieve or a piece of wire screen shaped like a cone, in the funnel and layer it with a piece of medical gauze.</li>
 <li>Fill up the funnel with tap water and ensure that the whole set-up does not leak.</li>
 <li>Scoop 20-30 g of fresh stool onto the middle of the medical gauze and ensure that it is completely submerged in water. Add more water if necessary. Fold the medical gauze over the sample.</li>
 <li>From below, shine artificial light at the funnel for 2 hr.</li>
 <li>Drain 50 milliliters (ml) of the lower portion of the water in the funnel into a centrifuge tube by gently releasing the clip on the rubber tube.</li>
 <li>Centrifuge the collected water at 600 x g (relative centrifugal force) for 5 min and carefully pour out the supernatant without disturbing the sediment. Retain the last few drops in the tube.</li>
 <li>Re-suspend the sediment and pipette 2 drops of the solution onto a microscope slide and examine the slide under a light microscope (40x magnification for detection and 100-400x magnification for species confirmation). First-stage larvae (L1) of S. stercoralis are alive and actively move under the microscope.</li>
</ol>
3. 20-m Shuttle Run Test
<ol>
 <li>Measure a 20-m flat and straight running course and mark it with cones.</li>
 <li>Help the test participant wear a heart rate monitor on the wrist and the corresponding transmitter around the chest18. This equipment allows the heart rate of the participant to be measured before and after the test and thus monitors whether the participant has exerted maximal effort.</li>
 <li>Instruct the participant to run back and forth on the course by following the pace of pre-recorded sound signals delimiting 20-m laps. Starting with a running speed of 8.5 kilometers per hour (km/hr), the frequency of the signals indicating 20-m intervals increases by an equivalent of 0.5 km/hr every min.</li>
 <li>Ensure that the participant starts a new 20-m interval each time a signal is sounded and finishes it before the next signal arrives.</li>
 <li>Stop the participant if he/she fails to follow the pace for two consecutive 20-m intervals.</li>
 <li>Record the highest running speed indicated for the last interval which the participant completed fully, along with the age and sex of the participant.</li>
 <li>Verify that the heart rate achieved at the end of the test is the child's maximal heart rate19. If this heart rate is not reached, the child might not have exerted maximal effort, and hence the test should be repeated.</li>
 <li>Estimate the maximum aerobic capacity (VO2 max) of the participant, using an equation put forth by L&eacute;ger and co-workers: VO2 max = 31.025 + 3.238 * speed (in km/hr) - 3.248 * age (in years) + 0.1536 * speed (in km/hr) * age (in years).</li>
</ol>
4. Representative Results
In terms of STH diagnosis with the Kato-Katz technique, unique characteristics of the different helminth eggs allow for the identification of most species20. The eggs of A.lumbricoides (approximately 45-75 micrometer (&mu;m) long and 35-50 &mu;m wide, Figure 1) have thicker shell walls than the eggs of T.trichiura (Figure 2) and hookworm (Figure 3). A fertilized egg of A.lumbricoides (Figure 1) has an outer albuminous coat, which makes it look golden brown, while a non-fertilized egg lacks this outer coat and is slightly elongated and larger overall. The eggs of T.trichiura (approximately 50 &mu;m long and 22 &mu;m wide, Figure 2) are elongated and have distinct polar plugs. Hookworm eggs (approximately 64-76 &mu;m long and 36-40 &mu;m wide, Figure 3), on the other hand, have a very thin wall, followed by a clear ring around a dense cluster of cells in the center of the egg.
S.stercoralis L1 larvae (Figure 4) are approximately 180-380 &mu;m long and have a very short buccal cavity. A distinct genital primordium (highlighted with a blue arrow in Figure 4) can be observed and the tail of the larvae (highlighted with a red arrow in Figure 4) has a sharp end.
As the true impact of STH infections on the physical fitness of children is still being debated13,14,15, there is no representative result for their impact at this point. Interpretation of 20-m shuttle run test results can be performed in various ways. The physical fitness of children can be compared between infected and non-infected subjects, who are similar in terms of age and sex, other potentially relevant health conditions, socioeconomic status, and cultural habits. This can be done in a cross-sectional survey13,14,15 where the STH infection status and physical fitness of infected and non-infected children are assessed at a particular time point. The strength of the evidence will be higher when following a randomized controlled trial design where the physical fitness of school-aged children is assessed before and after anthelminthic/placebo treatment, allowing for both short- and long-term evaluation. One can further try to identify a possible dose-response relationship between the intensity of STH infections and physical fitness and compare different levels of multiparasitism1 with physical fitness. In the work presented by Yap and co-workers15, physical fitness, as depicted by VO2 max, was found to be significantly reduced in children infected with T.trichiura compared to children who were not infected with T.trichiura (Figure 5).
<img alt="Figure 1" src="/files/ftp_upload/3966/3966fig1.jpg" /> 
Figure 1. Microscopic images of A.lumbricoides eggs: fertilized (A) and non-fertilized (B) under 500x magnification. Scale bar = 50 &mu;m.
<img alt="Figure 2" src="/files/ftp_upload/3966/3966fig2.jpg" /> 
Figure 2. Microscopic image of a T.trichiura egg under 500x magnification. Scale bar = 50 &mu;m.
<img alt="Figure 3" src="/files/ftp_upload/3966/3966fig3.jpg" /> 
Figure 3. Microscopic image of a hookworm egg under 500x magnification. Scale bar = 50 &mu;m.
<img alt="Figure 4" src="/files/ftp_upload/3966/3966fig4.jpg" /> 
Figure 4. Microscopic image of a S.stercoralis L1 larva under 250x magnification. Arrows indicate tail (red) and genital primordium (blue). Scale bar = 100 &mu;m.
<img alt="Figure 5" src="/files/ftp_upload/3966/3966fig5.jpg" /> 
Figure 5. Graph showing the physical fitness level of children infected with T.trichiura compared to the physical fitness level of children not infected with T. trichiura.

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K., Utzinger, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnosis,+clinical+features,+and+self-reported+morbidity+of+Strongyloides+stercoralis+and+hookworm+infection+in+a+co-endemic+setting.">Diagnosis, clinical features, and self-reported morbidity of Strongyloides stercoralis and hookworm infection in a co-endemic setting.</a> PLoS Neglected Tropical Diseases. 5, e1292 (2011).</li><li>World Health Organization. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Action+against+worms.">Action against worms.</a> WHO Newsletter. (11), (2008).</li><li>Montresor, A., Crompton, D. W. T., Hall, A., Bundy, D. A. 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M., Brigham, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improvements+in+physical+fitness+of+Kenyan+schoolboys+infected+with+hookworm,+Trichuris+trichiura+and+Ascaris+lumbricoides+following+a+single+dose+of+albendazole.">Improvements in physical fitness of Kenyan schoolboys infected with hookworm, Trichuris trichiura and Ascaris lumbricoides following a single dose of albendazole.</a> Transactions of the Royal Society of Tropical Medicine and Hygiene. 84, 277-282 (1990).</li><li>Stephenson, L. S., Latham, M. C., Adams, E. J., Kinoti, S. N., Pertet, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physical+fitness,+growth+and+appetite+of+Kenyan+school+boys+with+hookworm,+Trichuris+trichiura+and+Ascaris+lumbricoides+infections+are+improved+four+months+after+a+single+dose+of+albendazole.">Physical fitness, growth and appetite of Kenyan school boys with hookworm, Trichuris trichiura and Ascaris lumbricoides infections are improved four months after a single dose of albendazole.</a> Journal of Nutrition. 123, 1036-1046 (1993).</li><li>Bustinduy, A. L., Thomas, C. L., Fiutem, J. J., Parraga, I. M., Mungai, P. L., Muchiri, E. M., Mutuku, F., Kitron, U., King, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+fitness+of+Kenyan+children+with+polyparasitic+infections+using+the+20-meter+shuttle+run+test+as+a+morbidity+metric.">Measuring fitness of Kenyan children with polyparasitic infections using the 20-meter shuttle run test as a morbidity metric.</a> PLoS Neglected Tropical Diseases. 5, e1213 (2011).</li><li>M&#252;ller, I., Coulibaly, J. T., F&#252;rst, T., Knopp, S., Hattendorf, J., Krauth, S. J., Stete, K., Righetti, A. A., Glinz, D., Yao, A. K., P&#252;hse, U., N'Goran, E. 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N., Utzinger, J., Steinmann, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Soil-transmitted+helminth+infections+and+physical+fitness+in+school-aged+Bulang+children+of+southwest+China:+results+from+a+cross-sectional+survey.">Soil-transmitted helminth infections and physical fitness in school-aged Bulang children of southwest China: results from a cross-sectional survey.</a> Parasites and Vectors. 5, 50 (2012).</li><li>L&#233;ger, L. A., Mercier, D., Gadoury, C., Lambert, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+multistage+20+metre+shuttle+run+test+for+aerobic+fitness.">The multistage 20 metre shuttle run test for aerobic fitness.</a> Journal of Sports Sciences. 6, 93-101 (1988).</li><li>Tomkinson, G. R., L&#233;ger, L. A., Olds, T. 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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+Kato+thick-smear+technique+for+quantitative+diagnosis+of+helminth+infections.">Evaluation of Kato thick-smear technique for quantitative diagnosis of helminth infections.</a> American Journal of Tropical Medicine and Hygiene. 17, 382-391 (1968).</li><li>Dacombe, R. J., Crampin, A. C., Floyd, S., Randall, A., Ndhlovu, R., Bickle, Q., Fine, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time+delays+between+patient+and+laboratory+selectively+affect+accuracy+of+helminth+diagnosis.">Time delays between patient and laboratory selectively affect accuracy of helminth diagnosis.</a> Transactions of the Royal Society of Tropical Medicine and Hygiene. 101, 140-145 (2007).</li><li>Knopp, S., Mgeni, A. F., Khamis, I. S., Steinmann, P., Stothard, J. 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No conflicts of interest declared.

The three protocols described in this paper have been tested and executed in the south-western part of Yunnan province, People's Republic of China among members of the Bulang ethnic minority21 and in different parts of Africa6,13,14.
There are several templates available for the Kato-Katz technique. Each distinct hole-size will deliver a distinct amount of fecal material. For the calculation of EPGs, it is therefore necessary to know the unit of the template, so as to mul...

Note: Materials in this list are generic and can be obtained from different sources.
<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments </td>
		</tr>
		<tr>
			<td>Kato-Katz kit 
			[400 plastic templates with a hole of 6 millimeter (mm) (diameter) in a 1.5 mm thick template; 400 plastic spatula; a 20-m role of nylon screen of 80 mesh size; a 20-m role of hydrophilic cellophane, 34 &mu;m thick.]</td>
			<td>Vestergaard Frandsen</td>
			<td>&nbsp;</td>
			<td>Please note that the glycerin-malachite green solution is not included in this kit but can be bought from any chemical supplier.</td>
		</tr>
		<tr>
			<td>Glass funnel [about 8 centimeter (cm) wide and 6 cm deep]</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>The glass funnel for the Baermann technique should be able to hold about 60 mL of water. The size of the rubber hose and gauze is dependent on the type of glass funnel used.</td>
		</tr>
		<tr>
			<td>Biological microscope</td>
			<td>Olympus</td>
			<td>CX21LED/CX21</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Shanghai Medical Instruments Group</td>
			<td>80 - 2T</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Pre-recorded signals</td>
			<td>Bitworks Design</td>
			<td>Team Bleep Test Version 1.3.1</td>
			<td>iPhone application</td>
		</tr>
		<tr>
			<td>Heart rate monitor</td>
			<td>POLAR</td>
			<td>FT1</td>
			<td>Watch and transmitter</td>
		</tr>
	</tbody>
</table>

determining soil-transmitted helminth infection status and physical fitness of school-aged children

Soil-transmitted helminth (STH) infections are common. Indeed, more than 1 billion people are affected, mainly in the developing world where poverty prevails and hygiene behavior, water supply, and sanitation are often deficient1,2. Ascaris lumbricoides, Trichuris trichiura, and the two hookworm species, Ancylostoma duodenale and Necator americanus, are the most prevalent STHs3. The estimated global burden due to hookworm disease, ascariasis, and trichuriasis is 22.1, 10.5, and 6.4 million disability-adjusted life years (DALYs), respectively4. Furthermore, an estimated 30-100 million people are infected with Strongyloides stercoralis, the most neglected STH species of global significance which arguably also causes a considerable public health impact5,6. Multiple-species infections (i.e., different STHs harbored in a single individual) are common, and infections have been linked to lowered productivity and thus economic outlook of developing countries1,3.
For the diagnosis of common STHs, the World Health Organization (WHO) recommends the Kato-Katz technique7,8, which is a relatively straightforward method for determining the prevalence and intensity of such infections. It facilitates the detection of parasite eggs that infected subjects pass in their feces.
With regard to the diagnosis of S.stercoralis, there is currently no simple and accurate tool available. The Baermann technique is the most widely employed method for its diagnosis. The principle behind the Baermann technique is that active S.stercoralis larvae migrate out of an illuminated fresh fecal sample as the larvae are phototactic9. It requires less sophisticated laboratory materials and is less time consuming than culture and immunological methods5.
Morbidities associated with STH infections range from acute but common symptoms, such as abdominal pain, diarrhea, and pruritus, to chronic symptoms, such as anemia, under- and malnutrition, and cognitive impairment10. Since the symptoms are generally unspecific and subtle, they often go unnoticed, are considered a normal condition by affected individuals, or are treated as symptoms of other diseases that might be more common in a given setting. Hence, it is conceivable that the true burden of STH infections is underestimated by assessment tools relying on self-declared signs and symptoms as is usually the case in population-based surveys.
In the late 1980s and early 1990s, Stephenson and colleagues highlighted the possibility of STH infections lowering the physical fitness of boys aged 6-12 years11,12. This line of scientific inquiry gained new momentum recently13,14,15. The 20-meter (m) shuttle run test was developed and validated by L&eacute;ger et al.16 and is used worldwide to measure the aerobic fitness of children17. The test is easy to standardize and can be performed wherever a 20-m long and flat running course and an audio source are available, making its use attractive in resource-constrained settings13. To facilitate and standardize attempts at assessing whether STH infections have an effect on the physical fitness of school-aged children, we present methodologies that diagnose STH infections or measure physical fitness that are simple to execute and yet, provide accurate and reproducible outcomes. This will help to generate new evidence regarding the health impact of STH infections.

Watch this Scientific Journal Video about Determining Soil-transmitted Helminth Infection Status and Physical Fitness of School-aged Children at JoVE.com

Determining Soil-transmitted Helminth Infection Status and Physical Fitness of School-aged Children

Chronic infection with soil-transmitted helminths (STHs) causes malabsorption, stunting, and wasting in the growing child. Hence, it is plausible that these infections also reduce the physical fitness of children. Here, we visualize two techniques for the diagnosis of STHs and the 20-meter shuttle run test for assessing children's physical fitness.

Soil-transmitted helminth (STH) infections are common. Indeed, more than 1 billion people are affected, mainly in the developing world where poverty ...

determining-soil-transmitted-helminth-infection-status-physical

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JoVE Journal

Immunology and Infection

23.3K Views.  Swiss Tropical and Public Health Institute, Basel, Switzerland. Chronic infection with soil-transmitted helminths (STHs) causes malabsorption, stunting, and wasting in the growing child. Hence, it is plausible that these infections also reduce the physical fitness of children. Here, we visualize two techniques for the diagnosis of STHs and the 20-meter shuttle run test for assessing children's physical fitness.

Video: Determining Soil-transmitted Helminth Infection Status and Physical Fitness of School-aged Children

Determining the Phagocytic Activity of Clinical Antibody Samples

Antibody-driven phagocytosis is induced via the engagement of Fc receptors on professional phagocytes, and can contribute to both clearance as well as pathology of disease. While the properties of the variable domains of antibodies have long been considered critical to in vivo function, the ability of antibodies to recruit innate immune cells via their Fc domains has become increasingly appreciated as a major factor in their efficacy, both in the setting of recombinant monoclonal antibody therapy, as well as in the course of natural infection or vaccination1-3.
Importantly, despite its nomenclature as a constant domain, the antibody Fc domain does not have constant function, and is strongly modulated by IgG subclass (IgG1-4) and glycosylation at Asparagine 2974-6. Thus, this method to study functional differences of antigen-specific antibodies in clinical samples will facilitate correlation of the phagocytic potential of antibodies to disease state, susceptibility to infection, progression, or clinical outcome.
Furthermore, this effector function is particularly important in light of the documented ability of antibodies to enhance infection by providing pathogens access into host cells via Fc receptor-driven phagocytosis7. Additionally, there is some evidence that phagocytic uptake of immune complexes can impact the Th1/Th2 polarization of the immune response8.
Here, we describe an assay designed to detect differences in antibody-induced phagocytosis, which may be caused by differential IgG subclass, glycan structure at Asn297, as well as the ability to form immune complexes of antigen-specific antibodies in a high-throughput fashion. To this end, 1 &mu;m fluorescent beads are coated with antigen, then incubated with clinical antibody samples, generating fluorescent antigen specific immune complexes. These antibody-opsonized beads are then incubated with a monocytic cell line expressing multiple Fc&gamma;Rs, including both inhibitory and activating. Assay output can include phagocytic activity, cytokine secretion, and patterns of Fc&gamma;Rs usage, and are determined in a standardized manner, making this a highly useful system for parsing differences in this antibody-dependent effector function in both infection and vaccine-mediated protection9.

We present a high-throughput flow cytometric assay to determine the phagocytic activity of antigen-specific antibodies from clinical samples, utilizing fluorescent antigen-coated beads and a monocytic cell line expressing multiple Fc receptors&#x2014;providing receptor usage and phagocytic activity determinations in a standardized and reproducible fashion for any antigen of interest.

Antibody-driven phagocytosis is induced via the engagement of Fc receptors on professional phagocytes, and can contribute to both clearance as well as ...

Using Eggs from Schistosoma mansoni as an In vivo Model of Helminth-induced Lung Inflammation

Schistosoma parasites are blood flukes that infect an estimated 200 million people worldwide 1. In chronic infection with Schistosoma, the severe pathology, including liver fibrosis and splenomegaly, is caused by the immune response to the parasite eggs rather than the parasite itself 2. Parasite eggs induce a Th2 response characterized by the production of IL-4, IL-5 and IL-13, the alternative activation of macrophages and the recruitment of eosinophils. Here, we describe injection of Schistosoma mansoni eggs as a model to examine parasite-specific Th2 cytokine responses in the lung and draining lymph nodes, the formation of pulmonary granulomas surrounding the egg, and airway inflammation. 
Following intraperitoneal sensitization and intravenous challenge, S. mansoni eggs are transported to the lung via the pulmonary arteries where they are trapped within the lung parenchyma by granulomas composed of lymphocytes, eosinophils and alternatively activated macrophages 3-6. Associated with granuloma formation, inflammation in the broncho-alveolar spaces, expansion of the draining lymph nodes and CD4 T cell activation can be observed. Here we detail the protocol for isolating Schistosoma mansoni eggs from infected livers (modified from 7), sensitizing and challenging mice, and recovering the organs (broncho-alveolar lavage (BAL), lung and draining lymph nodes) for analysis. We also include representative histologic and immunologic data and suggestions for additional immunologic analysis. 
Overall, this method provides an in vivo model to investigate helminth-induced immunologic responses in the lung, which is broadly applicable to the study of Th2 inflammatory diseases including helminth infection, fibrotic diseases, allergic inflammation and asthma. Advantages of this model for the study of type 2 inflammation in the lung include the reproducibility of a potent Th2 inflammatory response in the lung and draining lymph nodes, the ease of assessment of inflammation by histologic examination of the granulomas surrounding the egg, and the potential for long-term storage of the parasite eggs.

Using Eggs from Schistosoma mansoni as an In vivo Model of Helminth-induced Lung Inflammation

Schistosoma mansoni eggs are potent stimulators of the T helper type 2 (Th2) immune response, characteristic of parasite infection, asthma and allergic inflammation. This protocol utilizes S. mansoni egg injection to generate a CD4 Th2 cytokine-induced inflammatory response in the lung, characterized by lung granuloma formation around the egg, eosinophilia and macrophage alternative activation.

Schistosoma parasites are blood flukes that infect an estimated 200 million people worldwide 1. In chronic infection with Schistosoma, the severe ...

Analysis of Pulmonary Dendritic Cell Maturation and Migration during Allergic Airway Inflammation

Dendritic cells (DCs) are the key players involved in initiation of adaptive immune response by activating antigen-specific T cells. DCs are present in peripheral tissues in steady state; however in response to antigen stimulation, DCs take up the antigen and rapidly migrate to the draining lymph nodes where they initiate T cell response against the antigen1,2. Additionally, DCs also play a key role in initiating autoimmune as well as allergic immune response3.
DCs play an essential role in both initiation of immune response and induction of tolerance in the setting of lung environment4. Lung environment is largely tolerogenic, owing to the exposure to vast array of environmental antigens5. However, in some individuals there is a break in tolerance, which leads to induction of allergy and asthma. In this study, we describe a strategy, which can be used to monitor airway DC maturation and migration in response to the antigen used for sensitization. The measurement of airway DC maturation and migration allows for assessment of the kinetics of immune response during airway allergic inflammation and also assists in understanding the magnitude of the subsequent immune response along with the underlying mechanisms.
Our strategy is based on the use of ovalbumin as a sensitizing agent. Ovalbumin-induced allergic asthma is a widely used model to reproduce the airway eosinophilia, pulmonary inflammation and elevated IgE levels found during asthma6,7. After sensitization, mice are challenged by intranasal delivery of FITC labeled ovalbumin, which allows for specific labeling of airway DCs which uptake ovalbumin. Next, using several DC specific markers, we can assess the maturation of these DCs and can also assess their migration to the draining lymph nodes by employing flow cytometry.

We describe a strategy to monitor maturation and migration of pulmonary dendritic cells in response to ovalbumin in the setting of ovalbumin induced allergic airway inflammation. This strategy can be modified to assess migration of pulmonary dendritic cells in settings of infection.

Dendritic cells (DCs) are the key players involved in initiation of adaptive immune response by activating antigen-specific T cells. DCs are present in ...

Fluorescence Microscopy Methods for Determining the Viability of Bacteria in Association with Mammalian Cells

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. Current protocols to address these questions include colony count assays, gentamicin protection assays, and electron microscopy. Colony count and gentamicin protection assays only assess the viability of the entire bacterial population and are unable to determine individual bacterial viability. Electron microscopy can be used to determine the viability of individual bacteria and provide information regarding their localization in host cells. However, bacteria often display a range of electron densities, making assessment of viability difficult. This article outlines protocols for the use of fluorescent dyes that reveal the viability of individual bacteria inside and associated with host cells. These assays were developed originally to assess survival of Neisseria gonorrhoeae in primary human neutrophils, but should be applicable to any bacterium-host cell interaction. These protocols combine membrane-permeable fluorescent dyes (SYTO9 and 4&#39;,6-diamidino-2-phenylindole [DAPI]), which stain all bacteria, with membrane-impermeable fluorescent dyes (propidium iodide and SYTOX Green), which are only accessible to nonviable bacteria. Prior to eukaryotic cell permeabilization, an antibody or fluorescent reagent is added to identify extracellular bacteria. Thus these assays discriminate the viability of bacteria adherent to and inside eukaryotic cells. A protocol is also provided for using the viability dyes in combination with fluorescent antibodies to eukaryotic cell markers, in order to determine the subcellular localization of individual bacteria. The bacterial viability dyes discussed in this article are a sensitive complement and/or alternative to traditional microbiology techniques to evaluate the viability of individual bacteria and provide information regarding where bacteria survive in host cells.

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. This article outlines protocols for the use of fluorescent dyes that reveal the viability of individual bacteria inside and associated with host cells.

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. Current protocols ...

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

The investigation of the intracellular protein levels of bacterial species is of importance to understanding the pathogenic mechanisms of diseases caused by these organisms. Here we describe a procedure for protein extraction from Burkholderia species based on mechanical lysis using glass beads in the presence of ethylenediamine tetraacetic acid and phenylmethylsulfonyl fluoride in phosphate buffered saline. This method can be used for different Burkholderia species, for different growth conditions, and it is likely suitable for the use in proteomic studies of other bacteria. Following protein extraction, a two-dimensional (2-D) gel electrophoresis proteomic technique is described to study global changes in the proteomes of these organisms. This method consists of the separation of proteins according to their isoelectric point by isoelectric focusing in the first dimension, followed by separation on the basis of molecular weight by acrylamide gel electrophoresis in the second dimension. Visualization of separated proteins is carried out by silver staining.

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

Members of the Burkholderia genus are pathogens of clinical importance. We describe a method for total bacterial protein extraction, using mechanical disruption, and 2-D gel electrophoresis for subsequent proteomic analysis.

The investigation of  the intracellular protein levels of bacterial species is of importance to  understanding the pathogenic mechanisms of diseases ...

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

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

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

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

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

Pairwise Growth Competition Assay for Determining the Replication Fitness of Human Immunodeficiency Viruses

In vitro fitness assays are essential tools for determining viral replication fitness for viruses such as HIV-1. Various measurements have been used to extrapolate viral replication fitness, ranging from the number of viral particles per infectious unit, growth rate in cell culture, and relative fitness derived from multiple-cycle growth competition assays. Growth competition assays provide a particularly sensitive measurement of fitness since the viruses are competing for cellular targets under identical growth conditions. There are several experimental factors to consider when conducting growth competition assays, including the multiplicity of infection (MOI), sampling times, and viral detection and fitness calculation methods. Each factor can affect the end result and hence must be considered carefully during the experimental design. The protocol presented here includes steps from constructing a new recombinant HIV-1 clone to performing growth competition assays and analyzing the experimental results. This protocol utilizes experimental parameter values previously shown to yield consistent and robust results. Alternatives are discussed, as some parameters need to be adjusted according to the cell type and viruses being studied. The protocol contains two alternative viral detection methods to provide flexibility as the availability of instruments, reagents and expertise varies between laboratories.

Growth competition between nearly isogenic viruses provides a sensitive measurement for determining relative replication fitness. The protocols described here include the construction of recombinant HIV-1 clones, virus propagation and growth competition and analysis methods optimized to yield sensitive and consistent results.

In vitro fitness assays are essential tools for determining viral replication fitness for viruses such as HIV-1. Various measurements have been used to ...

Determining Immune System Suppression versus CNS Protection for Pharmacological Interventions in Autoimmune Demyelination

A major hallmark of the autoimmune demyelinating disease multiple sclerosis (MS) is immune cell infiltration into the brain and spinal cord resulting in myelin destruction, which not only slows conduction of nerve impulses, but causes axonal injury resulting in motor and cognitive decline. Current treatments for MS focus on attenuating immune cell infiltration into the central nervous system (CNS). These treatments decrease the number of relapses, improving quality of life, but do not completely eliminate relapses so long-term disability is not improved. Therefore, therapeutic agents that protect the CNS are warranted. In both animal models as well as human patients with MS, T cell entry into the CNS is generally considered the initiating inflammatory event. In order to assess if a drug protects the CNS, any potential effects on immune cell infiltration or proliferation in the periphery must be ruled out. This protocol describes how to determine whether CNS protection observed after drug intervention is a consequence of attenuating CNS-infiltrating immune cells or blocking death of CNS cells during inflammatory insults. The ability to examine MS treatments that are protective to the CNS during inflammatory insults is highly critical for the advancement of therapeutic strategies since current treatments reduce, but do not completely eliminate, relapses (i.e., immune cell infiltration), leaving the CNS vulnerable to degeneration.

This protocol describes how to determine whether pharmacological treatments for experimental autoimmune encephalomyelitis show CNS protection as a consequence of suppressing immune cell infiltration or are neuroprotective during the onslaught of immune cell infiltration.

A major hallmark of the autoimmune demyelinating disease multiple sclerosis (MS) is immune cell infiltration into the brain and spinal cord resulting in ...

High-throughput Parallel Sequencing to Measure Fitness of Leptospira interrogans Transposon Insertion Mutants During Golden Syrian Hamster Infection

In this manuscript, we describe a transposon sequencing (Tn-Seq) technique to identify and quantify Leptospira interrogans mutants altered in fitness during infection of Golden Syrian hamsters. Tn-Seq combines random transposon mutagenesis with the power of high-throughput sequencing technology. Animals are challenged with a pool of transposon mutants (input pool), followed by harvesting of blood and tissues a few days later to identify and quantify the number of mutants in each organ (output pools). The output pools are compared to the input pool to evaluate the in vivo fitness of each mutant. This approach enables screening of a large pool of mutants in a limited number of animals. With minor modifications, this protocol can be performed with any animal model of leptospirosis, reservoir host models such as rats and acute infection models such as hamsters, as well as in vitro studies. Tn-Seq provides a powerful tool to screen for mutants with in vivo and in vitro fitness defects.

High-throughput Parallel Sequencing to Measure Fitness of Leptospira interrogans Transposon Insertion Mutants During Golden Syrian Hamster Infection

We describe here a technique that combines transposon mutagenesis with high-throughput sequencing to identify and quantify transposon leptospiral mutants in tissues after a challenge of hamsters. This protocol can be used to screen mutants for survival and dissemination in animals and can also be applied to in vitro studies.

In this manuscript, we describe a transposon sequencing (Tn-Seq) technique to identify and quantify Leptospira interrogans mutants altered in fitness ...

Immunization of Adult Zebrafish for the Preclinical Screening of DNA-based Vaccines

The interest in DNA-based vaccination has increased during the past two decades. DNA vaccination is based on the cloning of a sequence of a selected antigen or a combination of antigens into a plasmid, which enables a tailor-made and safe design. The administration of DNA vaccines into host cells leads to the expression of antigens that stimulate both humoral and cell-mediated immune responses. This report describes a protocol for the cloning of antigen sequences into the pCMV-EGFP plasmid, the immunization of adult zebrafish with the vaccine candidates by intramuscular microinjection, and the subsequent electroporation to improve intake. The vaccine antigens are expressed as green fluorescent protein (GFP)-fusion proteins, which allows the confirmation of the antigen expression under UV light from live fish and the quantification of expression levels of the fusion protein with ELISA, as well as their detection with a western blot analysis. The protective effect of the vaccine candidates is tested by infecting the fish with Mycobacterium marinum five weeks postvaccination, followed by the quantification of the bacteria with qPCR four weeks later. Compared to mammalian preclinical screening models, this method provides a cost-effective method for the preliminary screening of novel DNA-based vaccine candidates against a mycobacterial infection. The method can be further applied to screening DNA-based vaccines against various bacterial and viral diseases.

Here we describe a protocol for the immunization of the adult zebrafish (Danio rerio) with a DNA-based vaccine and demonstrate the validation of a successful vaccination event. This method is suitable for the preclinical screening of vaccine candidates in various infection models.

The interest in DNA-based vaccination has increased during the past two decades. DNA vaccination is based on the cloning of a sequence of a selected ...