Name: bioluminescence imaging to detect late stage infection of african trypanosomiasis
Uploaded: 2016-05-18T14:00:00
Description: Watch this Scientific Journal Video about Bioluminescence Imaging to Detect Late Stage Infection of African Trypanosomiasis at JoVE.com

We thank John Kelly and Martin Taylor (London School of Hygiene &#38; Tropical Medicine) for providing T. b. brucei GVR35-VSL-2 and Dr. Andrea Zelmer (LSHTM) for advice on in vivo imaging. This work was supported by the Bill and Melinda Gates Foundation Global Health Program (grant number OPPGH5337).

Ethics 
All work was carried out under the approval of the UK Home Office Animals (Scientific Procedures) Act 1986 and the London School of Hygiene &#38; Tropical Medicine Animal Welfare and Ethics Review Board. ARRIVE guideline are followed in this report.
1. In Vivo Passage of Bioluminescent Trypanosoma brucei brucei 
<ol>
	<li>Remove a cryopreserved stock (termed stabilate) of T. b. brucei strain GVR35-VSL-2 (available from Professor John Kelly at...

<ol>
	<li>Brun, R., Blum, J., Chappuis, F., Burri, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+African+trypanosomiasis.">Human African trypanosomiasis.</a> Lancet. 375 (9709), 148-159 (2010).</li><li>Rodgers, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trypanosomiasis+and+the+brain.">Trypanosomiasis and the brain.</a> Parasitology. 137 (14), 1995-2006 (2010).</li><li>Barrett, M. P., Boykin, D. W., Brun, R., Tidwell, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+African+trypanosomiasis:+pharmacological+re-engagement+with+a+neglected+disease.">Human African trypanosomiasis: pharmacological re-engagement with a neglected disease.</a> Br. J. Pharmacol. 152 (8), 1155-1171 (2007).</li><li>Barrett, M. P., Vincent, I. M., Burchmore, R. J. S., Kazibwe, A. J. N., Matovu, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drug+resistance+in+human+African+trypanosomiasis.">Drug resistance in human African trypanosomiasis.</a> Future Microbiol. 6 (9), 1037-1047 (2011).</li><li>Jennings, F. W., Whitelaw, D. D., Urquhart, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+relationship+between+duration+of+infection+with+Trypanosoma+brucei+in+mice+and+the+efficacy+of+chemotherapy.">The relationship between duration of infection with Trypanosoma brucei in mice and the efficacy of chemotherapy.</a> Parasitology. 75 (2), 143-153 (1977).</li><li>Andreu, N., Zelmer, A., Wiles, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+biophotonic+imaging+for+studies+of+infectious+disease.">Noninvasive biophotonic imaging for studies of infectious disease.</a> FEMS Microbiol. Rev. 35 (2), 360-394 (2011).</li><li>Sadikot, R. T., Blackwell, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioluminescence+imaging.">Bioluminescence imaging.</a> Proc. Am. Thorac. Soc. 2 (6), 537-540 (2005).</li><li>Burrell-Saward, H., Rodgers, J., Bradley, B., Croft, S. L., Ward, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+sensitive+and+reproducible+in+vivo+imaging+mouse+model+for+evaluation+of+drugs+against+late-stage+human+African+trypanosomiasis.">A sensitive and reproducible in vivo imaging mouse model for evaluation of drugs against late-stage human African trypanosomiasis.</a> J. Antimicrob. Chemother. 70 (2), 510-517 (2015).</li><li>McLatchie, A. P., 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=Highly+sensitive+in+vivo+imaging+of+Trypanosoma+brucei+expressing+'red-shifted'+luciferase.">Highly sensitive in vivo imaging of Trypanosoma brucei expressing 'red-shifted' luciferase.</a> PLoS Negl. Trop. Dis. 7 (11), e2571 (2013).</li><li>Jennings, F. W., 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=Human+African+trypanosomiasis:+potential+therapeutic+benefits+of+an+alternative+suramin+and+melarsoprol+regimen.">Human African trypanosomiasis: potential therapeutic benefits of an alternative suramin and melarsoprol regimen.</a> Parasitol. Int. 51 (4), 381-388 (2002).</li><li>Wiles, S., Robertson, B. D., Frankel, G., Kerton, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioluminescent+monitoring+of+in+vivo+colonization+and+clearance+dynamics+by+light-emitting+bacteria.">Bioluminescent monitoring of in vivo colonization and clearance dynamics by light-emitting bacteria.</a> Methods Mol. Biol. 574, 137-153 (2009).</li><li>Rodgers, J., Bradley, B., Kennedy, P. G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combination+chemotherapy+with+a+substance+P+receptor+antagonist+(aprepitant)+and+melarsoprol+in+a+mouse+model+of+human+African+trypanosomiasis.">Combination chemotherapy with a substance P receptor antagonist (aprepitant) and melarsoprol in a mouse model of human African trypanosomiasis.</a> Parasitol. Int. 56 (4), 321-324 (2007).</li><li>Kamerkar, S., Davis, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toxoplasma+on+the+brain:+understanding+host-pathogen+interactions+in+chronic+CNS+infection.">Toxoplasma on the brain: understanding host-pathogen interactions in chronic CNS infection.</a> J. Parasitol. Res. 2012, 589295 (2012).</li><li>Franke-Fayard, B., 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+malaria+parasite+sequestration:+CD36+is+the+major+receptor,+but+cerebral+pathology+is+unlinked+to+sequestration.">Murine malaria parasite sequestration: CD36 is the major receptor, but cerebral pathology is unlinked to sequestration.</a> Proc. Natl. Acad. Sci. U.S.A. 102 (32), 11468-11473 (2005).</li><li>Hitziger, N., Dellacasa, I., Albiger, B., Barragan, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissemination+of+Toxoplasma+gondii+to+immunoprivileged+organs+and+role+of+Toll/interleukin-1+receptor+signalling+for+host+resistance+assessed+by+in+vivo+bioluminescence+imaging.">Dissemination of Toxoplasma gondii to immunoprivileged organs and role of Toll/interleukin-1 receptor signalling for host resistance assessed by in vivo bioluminescence imaging.</a> Cell. Microbiol. 7 (6), 837-848 (2005).</li><li>Lewis, M. D., 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=Bioluminescence+imaging+of+chronic+Trypanosoma+cruzi+infections+reveals+tissue-specific+parasite+dynamics+and+heart+disease+in+the+absence+of+locally+persistent+infection.">Bioluminescence imaging of chronic Trypanosoma cruzi infections reveals tissue-specific parasite dynamics and heart disease in the absence of locally persistent infection.</a> Cell. Microbiol. 16 (9), 1285-1300 (2014).</li></ol>

The development of a bioluminescent T. b. brucei GVR35 strain allows the visualization of a trypanosome infection from the early to the late stage. Previous infection models were unable to detect the late stage, when parasites are in the brain, in real time from blood film microscopy, and required the culling and removal of brains from the infected mice to determine parasite burden12. The bioluminescence reduces inter-mouse variability as a single mouse can be tracked throughout the entirety of the in...

Human African trypanosomiasis (HAT), or sleeping sickness, is caused by the vector-borne protozoan parasites of the Trypanosoma brucei spp1. Estimated numbers of current cases is fewer than 7 thousand every year with almost 70 million people exposed to the risk of the parasite infection within the African continent. The disease, which is most often lethal if left untreated, comprises an early hemolymphatic stage where parasites are present in the blood, progressing to the late stage when parasites invade the central nervous system (CNS) and are no longer susceptible to treatment by early stage trypanosomal drugs2. The current drug therapies for late-stage HAT have both complex, prolonged, treatment regimens and severe adverse effects as well as reported resistance, therefore research into new drug therapies is imperative3,4. 
The study of late-stage human African trypanosomiasis (HAT) within traditional mouse models is lengthy and complex, with the removal of brain tissue being required to monitor parasitic burden5. The animal infective strain T. b. brucei is used as the study model of trypanosomiasis with the late stage appearing 21 days post infection (dpi). To monitor the wild type nonbioluminescent parasite infection in the mouse model, peripheral blood films or quantitative PCR are the only methods to determine parasite burden. For parasite burden in the brain, the mouse needs to be culled, brain excised and qPCR carried out on tissues, making it impossible to track parasites through multiple time points in the late stage infection. This results in the inability to follow real-time infections within the central nervous system (CNS). 
In vivo bioluminescence imaging (BLI) can provide highly sensitive, non-invasive detection of parasite dissemination and disease progression in a mouse model that can be followed in a single animal for the entirety of the experiment6. BLI is based on the emission of light in the visible spectrum produced by a luciferase-catalyzed reaction. The emitted photons are then detected by a charge coupled device (CCD) camera7. For this purpose, the pathogen is genetically modified to express a luciferase protein and the substrate, luciferin, is introduced at time points of interest by injection. The main advantage of this method is the ability to carry out longitudinal studies, in which the same animal can be imaged several times with minimal adverse effects. The acquired bioluminescence signal can be quantified, thus indicating the pathogen burden.
The optimization and validation of a red-shifted bioluminescent T. b. brucei has enabled the investigation of the late stage infection through non-invasive procedures, detecting parasites earlier than blood film microscopy and greatly reducing the time, cost and numbers of animals needed to study CNS infection and drug screening in late-stage trypanosomiasis8,9. In this protocol we demonstrate infection of mice with bioluminescent trypanosomes and how to then visualize the parasites in vivo for quantification of disease progression and CNS penetration.

<table border="0" cellpadding="0" cellspacing="0" width="860">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:233px;">PBS</td>
			<td style="width:189px;">Sigma, UK</td>
			<td style="width:155px;">P4417</td>
			<td style="width:283px;">tablets pH 7.4</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:233px;">Glucose</td>
			<td style="width:189px;">Sigma, UK</td>
			<td style="width:155px;">G8270</td>
			<td style="width:283px;">99.5% (molecular) grade</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:233px;">Ammonium chloride</td>
			<td style="width:189px;">Sigma, UK</td>
			<td style="width:155px;">A9434</td>
			<td style="width:283px;">99.5% (molecular) grade</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:233px;">Heparin (lithium salt)</td>
			<td style="width:189px;">Sigma, UK</td>
			<td style="width:155px;">H0878</td>
			<td style="width:283px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:233px;">Hi-FCS</td>
			<td style="width:189px;">Gibco, Life Technologies, UK</td>
			<td style="width:155px;">10500-064</td>
			<td style="width:283px;">500 ml</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:233px;">DPBS</td>
			<td style="width:189px;">Sigma, UK</td>
			<td style="width:155px;">D4031</td>
			<td style="width:283px;">Sterile filtered</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:233px;">Mr. Frosty</td>
			<td style="width:189px;">Nalgene, UK</td>
			<td style="width:155px;"></td>
			<td style="width:283px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:233px;">Giemsa</td>
			<td style="width:189px;">Sigma, UK</td>
			<td style="width:155px;">G5637</td>
			<td style="width:283px;"></td>
		</tr>
		<tr height="22">
			<td height="44" rowspan="2" style="height:44px;width:233px;">D-Luciferin</td>
			<td style="width:189px;">Perkin Elmer, UK</td>
			<td style="width:155px;"></td>
			<td style="width:283px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:189px;">Sigma, UK</td>
			<td style="width:155px;">115144-35-9</td>
			<td style="width:283px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:233px;">Diminazene aceturate</td>
			<td style="width:189px;">Sigma, UK</td>
			<td style="width:155px;">D7770</td>
			<td style="width:283px;">Analytical grade</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:233px;">IVIS Lumina II</td>
			<td style="width:189px;">Perkin Elmer, UK</td>
			<td style="width:155px;"></td>
			<td style="width:283px;">other bioimagers available e.g. from Bruker, Kodak</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:233px;">Living Image v. 4.2</td>
			<td style="width:189px;">Perkin Elmer, UK</td>
			<td style="width:155px;"></td>
			<td style="width:283px;">proprietary software for Perkin Elmer IVIS instruments; other instruments may have their own</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:233px;">1 ml syringe</td>
			<td style="width:189px;">Fisher Scientific, UK</td>
			<td>10142104</td>
			<td style="width:283px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:233px;">20 ml syringe</td>
			<td style="width:189px;">Fisher Scientific, UK</td>
			<td>10743785</td>
			<td style="width:283px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:233px;">25G Needles</td>
			<td style="width:189px;">Greiner Bio-one</td>
			<td>N2516</td>
			<td style="width:283px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:233px;">21G Needles</td>
			<td style="width:189px;">Greiner Bio-one</td>
			<td>N2138</td>
			<td style="width:283px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:233px;">Twin-frosted microscope slide</td>
			<td style="width:189px;">VWR, UK</td>
			<td>631-0117</td>
			<td style="width:283px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:233px;">1.5 ml microcentrifuge tube</td>
			<td style="width:189px;">StarLab, UK</td>
			<td>I1415-1000</td>
			<td style="width:283px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:233px;">7 ml Bijou tube</td>
			<td style="width:189px;">StarLab, UK</td>
			<td>E1412-0710</td>
			<td style="width:283px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:233px;">Mouse restrainer</td>
			<td style="width:189px;">Sigma, UK</td>
			<td>Z756903</td>
			<td style="width:283px;">our restrainer was made in-house, this is a similar model</td>
		</tr>
	</tbody>
</table>

bioluminescence imaging to detect late stage infection of african trypanosomiasis

Human African trypanosomiasis (HAT) is a multi-stage disease that manifests in two stages; an early blood stage and a late stage when the parasite invades the central nervous system (CNS). In vivo study of the late stage has been limited as traditional methodologies require the removal of the brain to determine the presence of the parasites.
Bioluminescence imaging is a non-invasive, highly sensitive form of optical imaging that enables the visualization of a luciferase-transfected pathogen in real-time. By using a transfected trypanosome strain that has the ability to produce late stage disease in mice we are able to study the kinetics of a CNS infection in a single animal throughout the course of infection, as well as observe the movement and dissemination of a systemic infection.
Here we describe a robust protocol to study CNS infections using a bioluminescence model of African trypanosomiasis, providing real time non-invasive observations which can be further analyzed with optional downstream approaches.

This protocol demonstrates how to follow disease progression following infection of mice with T. b. brucei, a model for human African trypanosomiasis. Figure 1 shows the timeline of the experimental protocol, demonstrating the timetable for treatment and imaging steps. Figure 2 demonstrates a typical field of view in a fixed Giemsa-stained blood smear used to quantitate peripheral parasitemia, with trypanosomes and red blood cells present. Develo...

Watch this Scientific Journal Video about Bioluminescence Imaging to Detect Late Stage Infection of African Trypanosomiasis at JoVE.com

Bioluminescence Imaging to Detect Late Stage Infection of African Trypanosomiasis

This manuscript describes the use of a bioluminescent strain of African trypanosomes to enable the tracking of late stage infection and demonstrates how in vivo live imaging can be used to visualize infections within the central nervous system in real-time.

Human African trypanosomiasis (HAT) is a multi-stage disease that manifests in two stages; an early blood stage and a late stage when the parasite invades ...

The overall goal of this protocol is to use in vivo live imaging to track a real-time trypanosome infection, visualizing the early blood to the late brain infection stages in the mouse, mimicking the clinical sleeping sickness progression. This method can help answer key questions in the trypanosome biology field, identifying potential new therapeutic treatments and evaluating the efficacy against the infection. The main advantage of this technique is that the drug evaluation can be established in 90 days, as opposed to the 180 days required for the previous role type model.This protocol can provide insight into human African trypanosomiasis but is also applicable to other models of infectious diseases such as tuberculosis or Chagas disease. Begin by placing 21 to 25 gram CD1 female mice into a heating box to dilate the tail veins. Then, place the warmed mice individually into a plastic restraining device and use a 25 gauge needle to inject 0.2 milliliters of diluted innoculum of the trypanosome brucei GVR35VSL2 intravenously into the tail vein, placing pressure at the injection site immediately after to stem the bleeding.To monitor the infection through blood-film microscopy, spot five micro-liters of blood onto a glass slide. Use a second glass slide to gently smear the drop across the first slide, producing a thin film. Allow the slides to air dry and then fix the blood in 100%methanol followed by 10 minutes in Giemsa.At the end of the incubation rinse the slides with distilled water and allow them to air dry. Then, count the number of trypanosomes in 10 fields of view under a 100x subjective. To track the infection by bio luminescent imaging warm an aliquot of D-luciferin to room temperature.Then, use a 25 gauge needle to intraperitoneally inject 150 milligrams per kilogram of the warmed luciferin per animal. Next, initialize the machine using the imager software according to the manufacturer's instructions. Then, when the camera and heated plate have reached the appropriate temperatures, place large black separators between the anesthetized animals to minimize the bleed-through of any bright bio luminescent signal.Image the mice using a series of exposure times with medium binning, one F-stop and an open filter and a field of view E of 12.5 times 12.5 centimeters. To ensure a thorough imaging of the mice, rotate the animals to obtain ventral, dorsal, and lateral views. On day 21 image and blood-film the mice as demonstrated.Then, dose the animals with 40 milligrams per kilogram of diminazine aceturate intraperitoneally to clear the peripheral parasitemia. After imaging the animals on day 35, place the excised brains onto a section of black plastic and pripet 50 micro-liters of 15 milligrams per milliliter of luciferin over the tissues. Then, image the brain using the same settings as demonstrated for the whole animal imaging.To quantify the degree of bio luminescence within specific regions of interest, or ROIs, select the ROI tool within the imaging software and create a rectangular ROI for the whole animal quantitation. Position the ROI over the mouse image from the tip of the nose to the base of the tail, and using the same size box for every animal and every time point, measure the total flux. To evaluate the ROI of the brain alone, create a circular ROI and place it over the animal's head region.Repeat for each animal and measure the total flux. Then, using the image with the highest bio luminescence and the image adjust tab on the tool palette, select a logarithmic scale and an appropriate color scale minimum and maximum for the image and apply this scale to all of the images within the experiment. The infection development can be following in vivo by sequential measurements of the bio luminescence to determine the degree of infection in the animals over the course of an experiment.As the infection progresses, an increase in the bio luminescence can be observed in all of the animals over the first 21 days as the signal becomes more disseminated and intense, with the highest regions of bio luminescence apparent in the spleen area. On day 21, the mice are treated with diminazine aceturate post-imaging, as just demonstrated, resulting in only low levels of bio luminescence present in the brain by day 28, as the peripheral infection is cleared. On day 35, the signal is not only still present in the head region, but has also become more intense, indicating a possible brain infection.Quantitation of both the peripheral parasitemia and bio luminescence allows the evaluation of the disease kinetics. For example, in this experiment, at day 7 a high bio luminescence signal of one times 10 to the 8th photons per second was observed, while the observed parasitemia was very low within the blood films generated on the same day. At day 28, the parasitemia became undetectable in the blood films due to the diminazene aceturate treatment administered on day 21, while the bio luminescence continued to remain above background levels until day 35, representing the bio luminescence observed in the head region and confirming the higher sensitivity of the bio luminescence analysis over the blood filming.Bio uminescence imaging of the brain itself reveals differences in the infection burden between animals, which does not appear to localize to specific anatomical regions of the tissue. While attempting this procedure it's important to remember to obtain a good range of exposure times to ensure quality imaging data, as overexposure, for example, negatively impacts important localization detail. Following this procedure, other downstream methods like quantitative PCR and immunohistochemistry can be performed to answer additional questions about brain parasite burden and parasite localization.After it's development, this technique paved the way for researchers in the field of trypanosome biology to perform drug evaluation within 90 days of infection, due to improved sensitivity of relapse detection. After watching this video, you should have a good understanding of how to use bio luminescence imaging of TB brucei infection to follow the disease progression from the blood to the late stage infection of the brain.

bioluminescence-imaging-to-detect-late-stage-infection-african

Research

JoVE Journal

Immunology and Infection

Comprehensive &amp; Cost Effective Laboratory Monitoring of HIV/AIDS: an African Role Model

We present the video about assisting anti-retroviral therapy (ART) by an apt laboratory service - representing a South-African role model for economical large scale diagnostic testing. In the low-income countries inexpensive ART has transformed the prospects for the survival of HIV seropositive patients but there are doubts whether there is a need for the laboratory monitoring of ART and at what costs - in situations when the overall quality of pathology services can still be very low. The appropriate answer is to establish economically sound services with better coordination and stricter internal quality assessment than seen in western countries. This video, photographed at location in the National Health Laboratory Services (NHLS-SA) at the Witwatersrand University, Johannesburg, South Africa, provides such a coordinated scheme expanding the original 2-color CD4-CD45 PanLeucoGating strategy (PLG). Thus the six modules of the video presentation reveal the simplicity of a 4-color flow cytometric assay to combine haematological, immunological and virology-related tests in a single tube. These video modules are: (i) the set-up of instruments; (ii) sample preparations; (iii) testing absolute counts and monitoring quality for each sample by bead-count-rate; (iv) the heamatological CD45 test for white cell counts and differentials; (v) the CD4 counts, and (vi) the activation of CD8+ T cells measured by CD38 display, a viral load related parameter. The potential cost-savings are remarkable. This arrangement is a prime example for the feasibility of performing &gt; 800-1000 tests per day with a stricter quality control than that applied in western laboratories, and also with a transfer of technology to other laboratories within a NHLS-SA network. Expert advisors, laboratory managers and policy makers who carry the duty of making decisions about introducing modern medical technology are frequently not in a position to see the latest technical details as carried out in the large regional laboratories with huge burdens of workload. Hence this video shows details of these new developments.

Anti-retroviral therapy to treat HIV/AIDS is monitored in South Africa on a large scale. Flow cytometry is combined for haematology (CD45), immunology (CD4) and viral-load linked CD38 assay. Recorded at NHLS-SA laboratories, Johannesburg, these modern methods are cost-efficient with heightened local internal quality control, serving as role-models for resource-limited diagnostics.

We present the video about assisting anti-retroviral therapy (ART) by an apt laboratory service - representing a South-African role model for economical ...

Bioluminescence Imaging of NADPH Oxidase Activity in Different Animal Models

NADPH oxidase is a critical enzyme that mediates antibacterial and antifungal host defense. In addition to its role in antimicrobial host defense, NADPH oxidase has critical signaling functions that modulate the inflammatory response 1. Thus, the development of a method to measure in "real-time" the kinetics of NADPH oxidase-derived ROS generation is expected to be a valuable research tool to understand mechanisms relevant to host defense, inflammation, and injury.
Chronic granulomatous disease (CGD) is an inherited disorder of the NADPH oxidase characterized by severe infections and excessive inflammation. Activation of the phagocyte NADPH oxidase requires translocation of its cytosolic subunits (p47phox, p67phox, and p40phox) and Rac to a membrane-bound flavocytochrome (composed of a gp91phox and p22phox heterodimer). Loss of function mutations in any of these NADPH oxidase components result in CGD. Similar to patients with CGD, gp91phox -deficient mice and p47phox-deficient mice have defective phagocyte NADPH oxidase activity and impaired host defense 2, 13. In addition to phagocytes, which contain the NADPH oxidase components described above, a variety of other cell types express different isoforms of NADPH oxidase.
Here, we describe a method to quantify ROS production in living mice and to delineate the contribution of NADPH oxidase to ROS generation in models of inflammation and injury. This method is based on ROS reacting with L-012 (an analogue of luminol) to emit luminescence that is recorded by a charge-coupled device (CCD). In the original description of the L-012 probe, L-012-dependent chemiluminescence was completely abolished by superoxide dismutase, indicating that the main ROS detected in this reaction was superoxide anion 14. Subsequent studies have shown that L-012 can detect other free radicals, including reactive nitrogen species 15, 16. Kielland et al. 16 showed that topical application of phorbol myristate acetate, a potent activator of NADPH oxidase, led to NADPH oxidase-dependent ROS generation that could be detected in mice using the luminescent probe L-012. In this model, they showed that L-012-dependent luminescence was abolished in p47phox-deficient mice.
We compared ROS generation in wildtype mice and NADPH oxidase-deficient p47phox-/- mice 2 in the following three models: 1) intratracheal administration of zymosan, a pro-inflammatory fungal cell wall-derived product that can activate NADPH oxidase; 2) cecal ligation and puncture (CLP), a model of intra-abdominal sepsis with secondary acute lung inflammation and injury; and 3) oral carbon tetrachloride (CCl4), a model of ROS-dependent hepatic injury. These models were specifically selected to evaluate NADPH oxidase-dependent ROS generation in the context of non-infectious inflammation, polymicrobial sepsis, and toxin-induced organ injury, respectively. Comparing bioluminescence in wildtype mice to p47phox-/- mice enables us to delineate the specific contribution of ROS generated by p47phox-containing NADPH oxidase to the bioluminescent signal in these models.
Bioluminescence imaging results that demonstrated increased ROS levels in wildtype mice compared to p47phox-/- mice indicated that NADPH oxidase is the major source of ROS generation in response to inflammatory stimuli. This method provides a minimally invasive approach for "real-time" monitoring of ROS generation during inflammation in vivo.

NADPH oxidase is the major source of reactive oxygen species (ROS) in phagocytes. Because of the ephemeral nature of ROS, it is difficult to measure and monitor ROS levels in living animals. A minimally invasive method for serial quantification of ROS in living mice is described.

NADPH oxidase is a critical enzyme that mediates antibacterial and antifungal host defense. In addition to its role in antimicrobial host defense, NADPH ...

Separation of Plasmodium falciparum Late Stage-infected Erythrocytes by Magnetic Means

Unlike other Plasmodium species, P. falciparum can be cultured in the lab, which facilitates its study 1. While the parasitemia achieved can reach the &asymp;40% limit, the investigator usually keeps the percentage at around 10%. In many cases it is necessary to isolate the parasite-containing red blood cells (RBCs) from the uninfected ones, to enrich the culture and proceed with a given experiment. 
When P. falciparum infects the erythrocyte, the parasite degrades and feeds from haemoglobin 2, 3. However, the parasite must deal with a very toxic iron-containing haem moiety 4, 5. The parasite eludes its toxicity by transforming the haem into an inert crystal polymer called haemozoin 6, 7. This iron-containing molecule is stored in its food vacuole and the metal in it has an oxidative state which differs from the one in haem 8. The ferric state of iron in the haemozoin confers on it a paramagnetic property absent in uninfected erythrocytes. As the invading parasite reaches maturity, the content of haemozoin also increases 9, which bestows even more paramagnetism on the latest stages of P. falciparum inside the erythrocyte.
Based on this paramagnetic property, the latest stages of P. falciparum infected-red blood cells can be separated by passing the culture through a column containing magnetic beads. These beads become magnetic when the columns containing them are placed on a magnet holder. Infected RBCs, due to their paramagnetism, will then be trapped inside the column, while the flow-through will contain, for the most part, uninfected erythrocytes and those containing early stages of the parasite.
Here, we describe the methodology to enrich the population of late stage parasites with magnetic columns, which maintains good parasite viability 10. After performing this procedure, the unattached culture can be returned to an incubator to allow the remaining parasites to continue growing.

Separation of Plasmodium falciparum Late Stage-infected Erythrocytes by Magnetic Means

The paramagnetic properties of hemozoin are used to isolate late stages of Plasmodium falciparum-infected red blood cells growing in culture. The method is simple and fast and does not affect the subsequent invasive capabilities of the parasites.

Unlike other Plasmodium species, P. falciparum can be cultured in the lab, which facilitates its study 1. While the parasitemia achieved can reach the ...

4D Multimodality Imaging of Citrobacter rodentium Infections in Mice

This protocol outlines the steps required to longitudinally monitor a bioluminescent bacterial infection using composite 3D diffuse light imaging tomography with integrated &mu;CT (DLIT-&mu;CT) and the subsequent use of this data to generate a four dimensional (4D) movie of the infection cycle. To develop the 4D infection movies and to validate the DLIT-&mu;CT imaging for bacterial infection studies using an IVIS Spectrum CT, we used infection with bioluminescent C. rodentium, which causes self-limiting colitis in mice. In this protocol, we outline the infection of mice with bioluminescent C. rodentium and non-invasive monitoring of colonization by daily DLIT-&mu;CT imaging and bacterial enumeration from feces for 8 days.
The use of the IVIS Spectrum CT facilitates seamless co-registration of optical and &mu;CT scans using a single imaging platform. The low dose &mu;CT modality enables the imaging of mice at multiple time points during infection, providing detailed anatomical localization of bioluminescent bacterial foci in 3D without causing artifacts from the cumulative radiation. Importantly, the 4D movies of infected mice provide a powerful analytical tool to monitor bacterial colonization dynamics in vivo.

4D Multimodality Imaging of Citrobacter rodentium Infections in Mice

Multi-modality imaging is a valuable approach for studying bacterial colonization in small animal models. This protocol outlines infection of mice with bioluminescent Citrobacter rodentium and the longitudinal monitoring of bacterial colonization using composite 3D diffuse light imaging tomography with &mu;CT imaging to create a 4D movie of C. rodentium infection.

This protocol outlines the steps required to  longitudinally monitor a bioluminescent bacterial infection using composite 3D diffuse light imaging ...

Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes

Bacterial intracellular pathogens can be conceived as molecular tools to dissect cellular signaling cascades due to their capacity to exquisitely manipulate and subvert cell functions which are required for the infection of host target tissues. Among these bacterial pathogens, Listeria monocytogenes is a Gram positive microorganism that has been used as a paradigm for intracellular parasitism in the characterization of cellular immune responses, and which has played instrumental roles in the discovery of molecular pathways controlling cytoskeletal and membrane trafficking dynamics. In this article, we describe a robust microscopical assay for the detection of late cellular infection stages of L. monocytogenes based on the fluorescent labeling of InlC, a secreted bacterial protein which accumulates in the cytoplasm of infected cells; this assay can be coupled to automated high-throughput small interfering RNA screens in order to characterize cellular signaling pathways involved in the up- or down-regulation of infection.

Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes

Listeria monocytogenes is a Gram positive bacterial pathogen frequently used as a major model for the study of intracellular parasitism. Imaging late L. monocytogenes infection stages within the context of small-interfering RNA screens allows for the global study of cellular pathways required for bacterial infection of target host cells.

Bacterial intracellular  pathogens can be conceived as molecular tools to dissect cellular signaling  cascades due to their capacity to exquisitely ...

Using Fluorescent Proteins to Monitor Glycosome Dynamics in the African Trypanosome

Trypanosoma brucei is a kinetoplastid parasite that causes human African trypanosomiasis (HAT), or sleeping sickness, and a wasting disease, nagana, in cattle1. The parasite alternates between the bloodstream of the mammalian host and the tsetse fly vector. The composition of many cellular organelles changes in response to these different extracellular conditions2-5.
Glycosomes are highly specialized peroxisomes in which many of the enzymes involved in glycolysis are compartmentalized. Glycosome composition changes in a developmental and environmentally regulated manner4-11. Currently, the most common techniques used to study glycosome dynamics are electron and fluorescence microscopy; techniques that are expensive, time and labor intensive, and not easily adapted to high throughput analyses.
To overcome these limitations, a fluorescent-glycosome reporter system in which enhanced yellow fluorescent protein (eYFP) is fused to a peroxisome targeting sequence (PTS2), which directs the fusion protein to glycosomes12, has been established. Upon import of the PTS2eYFP fusion protein, glycosomes become fluorescent. Organelle degradation and recycling results in the loss of fluorescence that can be measured by flow cytometry. Large numbers of cells (5,000 cells/sec) can be analyzed in real-time without extensive sample preparation such as fixation and mounting. This method offers a rapid way of detecting changes in organelle composition in response to fluctuating environmental conditions.

Glycosome dynamics in African trypanosomes are difficult to study by traditional cell biology techniques such as electron and fluorescence microscopy. As a means of observing dynamic organelle behavior, a fluorescent-organelle reporter system has been used in conjunction with flow cytometry to monitor real-time glycosome dynamics in live parasites.

Trypanosoma brucei is a kinetoplastid parasite that causes human African trypanosomiasis (HAT), or sleeping sickness, and a wasting disease, nagana, in ...

Mouse Fetal Liver Culture System to Dissect Target Gene Functions at the Early and Late Stages of Terminal Erythropoiesis

Erythropoiesis involves a dynamic process that begins with committed erythroid burst forming units (BFU-Es) followed by rapidly dividing erythroid colony forming units (CFU-Es). After CFU-Es, cells are morphologically recognizable and generally termed terminal erythroblasts. One of the challenges for the study of terminal erythropoiesis is the lack of experimental approaches to dissect gene functions in a chronological manner. In this protocol, we describe a unique strategy to determine gene functions in the early and late stages of terminal erythropoiesis. In this system, mouse fetal liver TER119 (mature erythroid cell marker) negative erythroblasts were purified and transduced with exogenous expression of cDNAs or small hairpin RNAs (shRNAs) for the genes of interest. The cells were subsequently cultured in medium containing growth factors other than erythropoietin (Epo) to maintain their progenitor stage for 12 hr while allowing the exogenous cDNAs or shRNAs to express. The cells were changed to Epo medium after 12 hr to induce cell differentiation and proliferation while the exogenous genetic materials were already expressed. This protocol facilitates analysis of gene functions in the early stage of terminal erythropoiesis. To study late stage terminal erythropoiesis, cells were immediately cultured in Epo medium after transduction. In this way, the cells were already differentiated to the late stage of terminal erythropoiesis when the transduced genetic materials were expressed. We recommend a general application of this strategy that would help understand detailed gene functions in different stages of terminal erythropoiesis.

We present an in vitro mouse fetal liver erythroblast culture system that dissects the early and late stages of terminal erythropoiesis. This system facilitates functional analysis of specific genes in different developmental stages.

Erythropoiesis involves a dynamic process that begins with committed erythroid burst forming units (BFU-Es) followed by rapidly dividing erythroid colony ...

In Vivo Investigation of Antimicrobial Blue Light Therapy for Multidrug-resistant Acinetobacter baumannii Burn Infections Using Bioluminescence Imaging

Burn infections continue to be an important cause of morbidity and mortality. The increasing emergence of multidrug-resistant (MDR) bacteria has led to the frequent failure of traditional antibiotic treatments. Alternative therapeutics are urgently needed to tackle MDR bacteria.
An innovative non-antibiotic approach, antimicrobial blue light (aBL), has shown promising effectiveness against MDR infections. The mechanism of action of aBL is not yet well understood. It is commonly hypothesized that naturally occurring endogenous photosensitizing chromophores in bacteria (e.g., iron-free porphyrins, flavins, etc.) are excited by aBL, which in turn produces cytotoxic reactive oxygen species (ROS) through a photochemical process.
Unlike another light-based antimicrobial approach, antimicrobial photodynamic therapy (aPDT), aBL therapy does not require the involvement of an exogenous photosensitizer. All it needs to take effect is the irradiation of blue light; therefore, it is simple and inexpensive. The aBL receptors are the endogenous cellular photosensitizers in bacteria, rather than the DNA. Thus, aBL is believed to be much less genotoxic to host cells than ultraviolet-C (UVC) irradiation, which directly causes DNA damage in host cells.
In this paper, we present a protocol to assess the effectiveness of aBL therapy for MDR Acinetobacter baumannii infections in a mouse model of burn injury. By using an engineered bioluminescent strain, we were able to noninvasively monitor the extent of infection in real time in living animals. This technique is also an effective tool for monitoring the spatial distribution of infections in animals.

In Vivo Investigation of Antimicrobial Blue Light Therapy for Multidrug-resistant Acinetobacter baumannii Burn Infections Using Bioluminescence Imaging

Infections caused by multidrug-resistant (MDR) bacterial strains have emerged as a serious threat to public health, necessitating the development of alternative therapeutics. We present a protocol to evaluate the effectiveness of antimicrobial blue light (aBL) therapy for MDR Acinetobacter baumannii infections in mouse burns by using bioluminescence imaging.

Burn infections continue to be an important cause of morbidity and mortality. The increasing emergence of multidrug-resistant (MDR) bacteria has led to ...

Imaging Ca2+ Responses During Shigella Infection of Epithelial Cells

Ca2+ is a ubiquitous ion involved in all known cellular processes. While global Ca2+ responses may affect cell fate, local variations in free Ca2+ cytosolic concentrations, linked to release from internal stores or an influx through plasma membrane channels, regulate cortical cell processes. Pathogens that adhere to or invade host cells trigger a reorganization of the actin cytoskeleton underlying the host plasma membrane, which likely affects both global and local Ca2+ signaling. Because these events may occur at low frequencies in a pseudo-stochastic manner over extended kinetics, the analysis of Ca2+ signals induced by pathogens raises major technical challenges that need to be addressed.
Here, we report protocols for the detection of global and local Ca2+ signals upon a Shigella infection of epithelial cells. In these protocols, artefacts linked to a prolonged exposure and photodamage associated with the excitation of Ca2+ fluorescent probes are troubleshot by stringently controlling the acquisition parameters over defined time periods during a Shigella invasion. Procedures are implemented to rigorously analyze the amplitude and frequency of global cytosolic Ca2+ signals during extended infection kinetics using the chemical probe Fluo-4.

Imaging Ca2+ Responses During Shigella Infection of Epithelial Cells

Here, we present protocols to visualize calcium (Ca2+) responses elicited by HeLa cells infected by Shigella. By optimizing the parameters of bacterial infection and imaging with Ca2+ fluorescent probes, atypical global and local Ca2+ signals induced by bacteria over a large range of infection kinetics are characterized.

Ca2+ is a ubiquitous ion involved in all known cellular processes. While global Ca2+ responses may affect cell fate, local variations in free Ca2+ ...

Purification of Extracellular Trypanosomes, Including African, from Blood by Anion-Exchangers (Diethylaminoethyl-cellulose Columns)

This method allows the separation of trypanosomes, parasites responsible for animal and human African trypanosomiasis (HAT), from infected blood. This is the best method for diagnosis of first stage HAT and furthermore this parasite purification method permits serological and research investigations.
HAT is caused by Tsetse fly transmitted Trypanosoma brucei gambiense and T. b. rhodesiense. Related trypanosomes are the causative agents of animal trypanosomiasis. Trypanosome detection is essential for HAT diagnosis, treatment and follow-up. The technique described here is the most sensitive parasite detection technique, adapted to field conditions for the diagnosis of T. b. gambiense HAT and can be completed within one hour. Blood is layered onto an anion-exchanger column (DEAE cellulose) previously adjusted to pH 8, and elution buffer is added. Highly negatively charged blood cells are adsorbed onto the column whereas the less negatively charged trypanosomes pass through. Collected trypanosomes are pelleted by centrifugation and observed by microscopy. Moreover, parasites are prepared without cellular damage whilst maintaining their infectivity.
Purified trypanosomes are required for immunological testing; they are used in the trypanolysis assay, the gold standard in HAT serology. Stained parasites are utilized in the card agglutination test (CATT) for field serology. Antigens from purified trypanosomes, such as variant surface glycoprotein, exoantigens, are also used in various immunoassays. The procedure described here is designed for African trypanosomes; consequently, chromatography conditions have to be adapted to each trypanosome strain, and more generally, to the blood of each species of host mammal.
These fascinating pathogens are easily purified and available to use in biochemical, molecular and cell biology studies including co-culture with host cells to investigate host-parasite relationships at the level of membrane receptors, signaling, and gene expression; drug testing in vitro; investigation of gene deletion, mutation, or overexpression on metabolic processes, cytoskeletal biogenesis and parasite survival.

Purification of Extracellular Trypanosomes, Including African, from Blood by Anion-Exchangers Diethylaminoethyl-cellulose Columns

This method of trypanosome separation from blood depends on their surface charge being less negative than mammalian blood cells. Infected blood is placed and treated on an anion-exchanger column. This method, the most fitting diagnostic for African trypanosomiasis, provides purified parasites for immunological, biological, biochemical, pharmaceutical and molecular biology investigations.

This method allows the separation of trypanosomes, parasites responsible for animal and human African trypanosomiasis (HAT), from infected blood. This is ...