Name: purification of extracellular trypanosomes, including african, from blood by anion-exchangers (diethylaminoethyl-cellulose columns)
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We thank all members of UMR 177 INTERTRYP IRD CIRAD Universit&#233; de Bordeaux. This research was supported by internal funding from University of Bordeaux and support from the ANR, LABEX ParaFrap ANR-11-LABX-0024, and from the Association pour le d&#233;veloppement de la recherche en parasitologie et m&#233;decine tropicale and the Service de coop&#233;ration et d&#39;action culturelle de l&#39;Ambassade de France &#224; Bangui (Centrafrique).

Investigations conformed to the Guidelines for the Care and Use of Laboratory Animals (NIH Publication No. 85&#177;23, revised 1996). Protocols were approved by our local ethics committee.
1. Animals
<ol>
	<li>Keep female Swiss (OF-1) mice aged eight to ten weeks old, 20-25 g, in an animal housing facility fifteen days before each experiment. House them in ventilated boxes that are kept in a protected, temperature (22 &#176;C) and humidity (50%) controlled room, with 12 hours on/off ...

<ol>
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A., Da Silva, W. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibody-dependent+cytotoxicity+against+Trypanosoma+cruzi.">Antibody-dependent cytotoxicity against Trypanosoma cruzi.</a> Parasitology. 75 (3), 317-323 (1977).</li><li>Herbert, W. J., Lumsden, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trypanosoma+brucei:+a+rapid+&amp;#34;matching&amp;#34;+method+for+estimating+the+host's+parasitemia.">Trypanosoma brucei: a rapid &amp;#34;matching&amp;#34; method for estimating the host's parasitemia.</a> Experimental Parasitology. 40 (3), 427-431 (1976).</li><li>Dauchy, F. A., 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=Trypanosoma+brucei+CYP51:+Essentiality+and+Targeting+Therapy+in+an+Experimental+Model.">Trypanosoma brucei CYP51: Essentiality and Targeting Therapy in an Experimental Model.</a> PLoS Neglected Tropical Diseases. 10 (11), e0005125 (2016).</li><li>Raz, B., Iten, M., Grether-B&#252;hler, Y., Kaminsky, R., Brun, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Alamar+Blue+assay+to+determine+drug+sensitivity+of+African+trypanosomes+(T.+b.+rhodesiense+and+T.+b.+gambiense)+in+vitro.">The Alamar Blue assay to determine drug sensitivity of African trypanosomes (T. b. rhodesiense and T. b. gambiense) in vitro.</a> Acta Tropica. 68 (2), 139-147 (1997).</li><li>Albright, J. W., Albright, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+growth+of+Trypanosoma+musculi+in+cell-free+medium+conditioned+by+rodent+macrophages+and+mercaptoethanol.">In vitro growth of Trypanosoma musculi in cell-free medium conditioned by rodent macrophages and mercaptoethanol.</a> International Journal for Parasitology. 10 (2), 137-142 (1980).</li><li>Gobert, 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=L-Arginine+availability+modulates+local+nitric+oxide+production+and+parasite+killing+in+experimental+trypanosomiasis.">L-Arginine availability modulates local nitric oxide production and parasite killing in experimental trypanosomiasis.</a> Infection and Immunity. 68 (8), 4653-4657 (2000).</li><li>De Muylder, G., 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=A+Trypanosoma+brucei+kinesin+heavy+chain+promotes+parasite+growth+by+triggering+host+arginase+activity.">A Trypanosoma brucei kinesin heavy chain promotes parasite growth by triggering host arginase activity.</a> PLoS Pathogens. 9 (10), e1003731 (2013).</li><li>Nzoumbou-Boko, R., 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=Trypanosoma+musculi+Infection+in+Mice+Critically+Relies+on+Mannose+Receptor-Mediated+Arginase+Induction+by+a+TbKHC1+Kinesin+H+Chain+Homolog.">Trypanosoma musculi Infection in Mice Critically Relies on Mannose Receptor-Mediated Arginase Induction by a TbKHC1 Kinesin H Chain Homolog.</a> Journal of Immunology. 199 (5), 1762-1771 (2017).</li><li>Bonhivers, M., Nowacki, S., Landrein, N., Robinson, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biogenesis+of+the+trypanosome+endo-exocytotic+organelle+is+cytoskeleton+mediated.">Biogenesis of the trypanosome endo-exocytotic organelle is cytoskeleton mediated.</a> PLoS Biology. 6 (5), e105 (2008).</li><li>Albisetti, A., 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=Interaction+between+the+flagellar+pocket+collar+and+the+hook+complex+via+a+novel+microtubule-binding+protein+in+Trypanosoma+brucei.">Interaction between the flagellar pocket collar and the hook complex via a novel microtubule-binding protein in Trypanosoma brucei.</a> PLoS Pathogens. 13 (11), e1006710 (2017).</li><li>Cross, G. A. M., Klein, R. A., Linstead, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Utilization+of+amino+acids+by+Trypanosoma+brucei+in+culture:+L-threonine+as+a+precursor+for+acetate.">Utilization of amino acids by Trypanosoma brucei in culture: L-threonine as a precursor for acetate.</a> Parasitology. 71 (2), 311-326 (1975).</li><li>Bringaud, F., Rivi&#232;re, L., Coustou, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Energy+metabolism+of+trypanosomatids:+adaptation+to+available+carbon+sources.">Energy metabolism of trypanosomatids: adaptation to available carbon sources.</a> Molecular and Biochemical Parasitology. 149 (1), 1-9 (2006).</li><li>Mazet, M., 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=Revisiting+the+central+metabolism+of+the+bloodstream+forms+of+Trypanosoma+brucei:+production+of+acetate+in+the+mitochondrion+is+essential+for+the+parasite+viability.">Revisiting the central metabolism of the bloodstream forms of Trypanosoma brucei: production of acetate in the mitochondrion is essential for the parasite viability.</a> PLoS Neglected Tropical Diseases. 7 (12), e2587 (2013).</li><li>Coutton, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutations+in+CFAP43+and+CFAP44+cause+male+infertility+and+flagellum+defects+in+Trypanosoma+and+human.">Mutations in CFAP43 and CFAP44 cause male infertility and flagellum defects in Trypanosoma and human.</a> Nature Communications. 9 (1), 686 (2018).</li><li>Cnops, J., Magez, S., De Trez, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Escape+mechanisms+of+African+trypanosomes:+Why+trypanosomosis+is+keeping+us+awake.">Escape mechanisms of African trypanosomes: Why trypanosomosis is keeping us awake.</a> Parasitology. 142 (3), 417-427 (2015).</li><li>Barrett, M. 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Purified trypanosomes represent a powerful means to study immunology, biochemistry, cellular and molecular biology. Large expanses of data and results have been obtained from trypanosomes, which has then helped to obtain information from other eukaryotic cells30. Trypanosomes are also the subject of important and interesting research because they have devised numerous mechanisms that permit them to survive and grow in two very different environments: the tsetse fly vector and the mammalian host<su...

The method presented described here allows the separation of trypanosomes, parasites responsible for animal and human African trypanosomiasis (HAT), from blood. This is the best method for diagnosis of first stage HAT and furthermore this parasite purification method permits robust serological and research investigation.
HAT is caused by Tsetse fly transmitted Trypanosoma brucei gambiense and T. b. rhodesiense1. These protozoan parasites multiply extracellularly in the bloodstream, lymph, and interstitial fluids during the first stage of the disease (hemolymphatic stage). The second stage (meningoencephalitic stage) begins when parasites cross the blood brain barrier; neurological signs, including a sleep disorder, which has given its name &#34;sleeping sickness&#34; to this disease, are typical of this second-stage2. Related trypanosomes (T. evansi, T. congolense, T. vivax, T. b. brucei) are the causative agents of animal African trypanosomosis (AAT)3.
The World Health Organization (WHO) aims to eliminate HAT as a public health problem by 2020 and to stop transmission by 20304. The recent introduction of rapid diagnosis tests has improved serological diagnosis1,4,5. Several molecular diagnostic tests have been developed but their role in field diagnostics has not yet been established5. They are used to identify the sub-species of the brucei group and atypical trypanosomiasis caused by parasites responsible for animal trypanosomosis6.
The detection of the parasite is essential for the diagnosis, treatment and follow-up, as serology can give false positive and unfortunately false negative results1. The direct microscopical observation of these hemoflagellate protists is difficult in HAT cases that are caused by T. b. gambiense, (more than 95% of cases) as low parasitemias are the rule, whereas for HAT caused by T. b. rhodesiense, a large number of parasites are frequently present in the blood. Various concentration techniques have been used, such as thick drop and capillary tube centrifugation (CTC), but the separation of parasites from blood by a column of anion-exchanger (DEAE cellulose) followed by centrifugation and microscopic observation of the pellet, is the most sensitive method (around 50 parasites/mL of blood can be detected)1,7. Consequently, the purification of trypanosomes by this anion-exchangers (DEAE cellulose) method is the best and, to date, the reference method for visualizing and isolating parasites from blood for HAT diagnosis. In field conditions, a mini-column of DEAE cellulose has been successfully used and several improvements have facilitated microscopical observation7,8.
The method of trypanosome separation from blood, described below, depends on parasite surface charge, which is less negative than mammalian blood cells9. Interestingly, this method was developed 50 years ago, in 1968 by Dr. Sheila Lanham, and remains the gold standard for detection and preparation of bloodstream trypanosomes. It is fast and reproducible for salivarian trypanosomes from a wide range of mammals, permitting the diagnosis of both animal and human trypanosomiasis10.
To obtain living, purified parasites, infected blood is added onto an anion-exchanger column. Chromatography conditions (mainly pH, ionic strength of buffers/media) have to be adapted to each trypanosome species, and more generally, to each mix of mammalian blood cells and trypanosomes10. Elution buffer is precisely adjusted to pH 8 for most African trypanosomes10. This method favors the concentration of parasites found in the blood of patients, because parasitemias can be too low to be detected by microscopic observation alone, and it also enables laboratory investigations. Working with freshly isolated trypanosomes and on blood from infected animals, using this technique, is more pertinent for various investigations than studies with parasites that have been cultured in axenic conditions in the laboratory for an indefinite period.
Host-parasite relationships are best studied with a parasite infecting its natural host, therefore, T. musculi, a natural murine parasite, which is representative of extracellular trypanosomes, has many advantages as murine infection involves in a small laboratory animal and does not require biohazard safety level (BSL) conditions. T. musculi does not kill immunocompetent mice, unlike many other Trypanosoma species, including human pathogens. T. musculi are not eliminated in T cell-deprived mice and parasitemias can be increased&#160;in infected mice by modifying food and nutrient intake11. This parasite modulates the immune response in co-infections with other pathogens12. T. musculi from infected mice exhibit differences from cultured T. musculi, for example, the expression of membrane Fc receptors is lost in T. musculi axenic cultures, compared to parasites purified from infected mice13,14. Excreted-secreted factors (ESF) are also qualitatively and quantitatively less expressed in axenic trypanosome cultures and differ between strains isolated in endemic areas15. ESF are the first antigens to be displayed to the host immune system and so play an important role in the initial host immune response16.
In experimentally infected animals for laboratory investigations, this protocol facilitates experimentation on a greater number of parasites, minimizing the number of mice required especially when using immunosuppressed animals. The variant surface glycoproteins (VSGs) that are used in the Card Agglutination Test for Trypanosomiasis (CATT) in mass screening are still purified from trypanosomes that are propagated in rats. The two rapid diagnostic tests (individually wrapped cassettes) that are now available for use in the field, are still using an infective model source of native VSGs and not in vitro cultured trypanosomes1,4,5. The advancement in the study of trypanosome immunology and biology has been facilitated since these DEAE cellulose purified parasites can be easily obtained in large quantities from naturally or experimentally infected hosts, and in particular, rodents.

<table border="0" cellpadding="0" cellspacing="0" style="width:1036px;" width="1036">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">10 mL Pipettes&#160;</td>
			<td style="width:381px;">Falcon</td>
			<td style="width:184px;">357,551</td>
			<td style="width:172px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">2 mL Pipettes&#160;</td>
			<td style="width:381px;">Falcon</td>
			<td style="width:184px;">352,507</td>
			<td style="width:172px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Centrifugation tube 50 mL</td>
			<td style="width:381px;">Falcon</td>
			<td style="width:184px;">352,070</td>
			<td style="width:172px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Centrifuge</td>
			<td style="width:381px;">Sigma Aldrich</td>
			<td style="width:184px;">4K15</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">DEAE cellulose</td>
			<td style="width:381px;">Santa Cruz</td>
			<td style="width:184px;">s/c- 211213</td>
			<td>100 G</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">filter paper&#160;</td>
			<td style="width:381px;">Whatman</td>
			<td style="width:184px;">1,001,125</td>
			<td style="width:172px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Flat bottom flask narrow neck</td>
			<td style="width:381px;">Duran</td>
			<td style="width:184px;">21 711 76</td>
			<td style="width:172px;">6000 mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Glucose&#160;</td>
			<td style="width:381px;">VWR</td>
			<td style="width:184px;">101174Y</td>
			<td>500 G</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Heparin</td>
			<td style="width:381px;">Sigma Aldrich</td>
			<td style="width:184px;">H3149-50KU</td>
			<td>5 000 U</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:299px;">KH2PO4</td>
			<td style="width:381px;">VWR</td>
			<td style="width:184px;">120 26936.260</td>
			<td>500 G</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:299px;">Microscope</td>
			<td style="width:381px;">Olympus</td>
			<td style="width:184px;">CH-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microscope coverslips</td>
			<td style="width:381px;">Thermofisher scientific</td>
			<td style="width:184px;">CB00100RA020MNT0</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Microscope slides</td>
			<td style="width:381px;">Thermofisher scientific</td>
			<td style="width:184px;">AGAA000001</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:299px;">Na2HPO4&#160;</td>
			<td style="width:381px;">VWR</td>
			<td style="width:184px;">100 28026;260</td>
			<td>500 G</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">NaCl</td>
			<td style="width:381px;">VWR</td>
			<td style="width:184px;">27800.291</td>
			<td>1 KG</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:299px;">NaH2PO4&#160;</td>
			<td style="width:381px;">VWR</td>
			<td style="width:184px;">110 33616;262</td>
			<td>500 G</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Nalgene Plastic Media Bottles size 125 mL</td>
			<td style="width:381px;">Thermofisher scientific</td>
			<td style="width:184px;">342024-0125</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Nalgene Plastic Media Bottles size 500 mL</td>
			<td style="width:381px;">Thermofisher scientific</td>
			<td style="width:184px;">342024-0500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Pasteur Pipette</td>
			<td style="width:381px;">VWR</td>
			<td style="width:184px;">BRND125400</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">Penicillin 10,000 UI/Streptomycin 10,000 &#181;g</td>
			<td style="width:381px;">EUROBIO</td>
			<td style="width:184px;">CABPES01 OU</td>
			<td>100 mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Phenol red</td>
			<td style="width:381px;">Sigma Aldrich</td>
			<td style="width:184px;">P0290</td>
			<td>100 mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Syringue&#160;</td>
			<td style="width:381px;">Dutscher</td>
			<td style="width:184px;">SS+10S21381</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Tissue culture hood</td>
			<td style="width:381px;">Thermoelectro Corporation</td>
			<td style="width:184px;">MSC-12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Trypanosoma brucei brucei</td>
			<td style="width:381px;">Institute of Tropical Medicine (Antwerp, Belgium).</td>
			<td style="width:184px;"></td>
			<td>ANTAT 1.1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Trypanosoma brucei gambiense</td>
			<td style="width:381px;">Institute of Tropical Medicine (Antwerp, Belgium).</td>
			<td style="width:184px;"></td>
			<td>ITMAP 1893</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Trypanosoma musculi</td>
			<td>London School of Hygiene and Tropical Medicine (UK)</td>
			<td style="width:184px;"></td>
			<td>Partinico II</td>
		</tr>
	</tbody>
</table>

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.

Purified trypanosomes have been used in pharmaceutical tests. Parasites are transferred into culture wells containing serial dilutions of specific drugs, either alone or mixed19. Microscopic observations, evaluating motility is a marker of viability, can be performed when only a few dugs are being tested, whereas AlamarBlue cell viability assay is an excellent method for large motility assays during drug screening20. The effect&#160;of penta...

Watch this Scientific Journal Video about Purification of Extracellular Trypanosomes, Including African, from Blood by Anion-Exchangers Diethylaminoethyl-cellulose Columns at JoVE.com

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.

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 ...

African trypanosomes are responsible for human African trypanosomiasis, or sleeping sickness and animal African trypanosomiasis. The trypanosomes that cause sleeping sickness are tsetse fly transmitted protist parasites present in many geographical foci in sub Saharan Africa. The detection of the parasite is essential for the diagnosis, treatment, and follow-up.A serology can give false positive and unfortunately, false negative results. To aid detection in the blood, trypanosomes have to be concentrated. Various parasite concentration techniques have been used.The most sensitive method is the separation of parasites from blood by a column of anion exchanger, DEAE cellulose followed by centrifugation and microscopic observation. Consequently, the DEAE cellulose method is the best and remains to date, the reference method for visualizing and isolating parasites from blood for human African trypanosomiasis diagnosis, especially in field conditions. This method, the most fitting diagnostic for African trypanosomiasis also provides purified parasites for immunological, biological, biochemical, pharmaceutical, and molecular biological investigations.All investigations conformed to the guide for the care and use of laboratory animals and agreement has been obtained from the French authorities and all the protocols used have been approved by our local ethics committee. Weigh out each substance according to the written protocol. Add distilled water and adjust precisely to pH 8 with potassium dihydrogen phosphate to make the following buffered solutions, concentrated phosphate buffer saline 2X.Add distilled water and glucose for phosphate buffered saline glucose. For 100 milliliters of elution buffer, add 100 units per milliliter of penicillin, 100 micrograms per milliliter of streptomycin, and five micrograms milliliter of phenol red to phosphate buffered saline glucose. 100 grams of DEAE cellulose is first washed with three liters of distilled water and then left to settle.Fine particles are discarded. The washes are repeated until the supernatant is clear. Concentrated phosphate buffered saline 2X is added and the DEAE cellulose is stirred.The pH is adjusted to eight with one molar potassium phosphate. The cellulose is then washed twice with distilled water and twice with phosphate buffered saline glucose. The DEAE cellulose and phosphate buffered saline glucose are mixed in equal volumes.Alloy quartered and stored at room temperature or four degrees celsius or at minus 20 degrees celsius for prolonged storage. Trypanosome infected blood is stored in liquid nitrogen. An alloy quart of one milliliter is thawed at room temperature.Parasites are carefully collected from the cryotube using a needle and a one milliliter syringe. The viability of thawed parasites is assessed by the motility as visualized by light microscopy observation before infection. Parasites are injected into the peritoneal cavities of two mice, 500 microliters per mouse.Each day post infection, parasitemia is assessed by collecting a drop of blood from the tail with a needle and checked by microscopy. When the parasitemia is at a suitable level, infected blood is collected. A 10 milliliter syringe is supported in a clamp or holder and a precut piece of filter paper is added.Prepared DEAE cellulose is poured into the syringe up to the eight or nine milliliter level. The column is washed with the elution buffer. Two milliliters of infected blood is carefully placed on top of the column and trypanosomes are collected in the column eluate in a 50 milliliter centrifugation tube.Elution buffer is regularly added according to the transit of trypanosomes. The transit of trypanosomes through the column is checked by taking a drop of column eluate at regular intervals and observing for trypanosomes using light microscopy. When trypanosomes are no longer observed in the eluate, the conical tube is centrifuged at 1, 800 times G for 10 minutes at four degrees celsius.The supernatant is discarded and trypanosomes in the pallet are counted with a hemocytometer. Collected trypanosomes are subsequently diluted in the medium required for the planned investigation. This method, the most fitting diagnostic for African trypanosomiasis provides purified parasites for immunological, biological, biochemical, pharmaceutical, and molecular biological investigations.These studies have been developed because DEAE cellulose purified parasites can be easily obtained in large quantities from naturally or experimentally infected hosts and in particular, rodents. Trypanosome viability in the presence of pharmaceutical agents is assessed by microscopic observation. Parasite viability is associated with motility and therefore, can be checked by eye under a light microscope at 40 times or higher magnification.Parasite viability is assessed by measuring the percentage of motile forms. Dilutions of test compounds are added into each well of a 96 well tissue culture plate while control wells are mock treated. Each concentration is assessed in triplicate.The number of viable parasites for each dilution is compared to the number of parasites in control wells. The effects of Pentamidine, a reference drug, used in human African trypanosomiasis therapy is displayed in this figure. Trypanosome macrophage co-cultures allow the investigation of host parasite interaction at the cellular level.In macrophage parasite co-cultures, extra cellular trypanosomes induce production of macrophage arginase that hydrolyzes arginine to ornithine. Ornithine is the precursor of polyamines and trypanothione, essential for parasite survival and growth. Furthermore, arginine depletion decreases the production of trypanocidal nitric oxide.Subsequently, addition of an arginase inhibitor, S-L-cysteine to co-cultures dramatically reduces macrophage induced parasite growth. Arginase induction is mediated by trypanosome excreted and or secreted factors. This arginase inducing factor has been identified as an orthine kinesin.The kinesin binds to C-type lectin mannose binding receptors. Arginase is induced and produces ornithine which favors parasite growth. Arginase is not induced by kinesin in macrophages cultured with 12.5 millimolar mannose or in macrophages from mice where the macrophage mannose receptors have been deleted.Further examples of cell biology and biochemical experimentation using DAEA cellulose purified trypanosomes are demonstrated in studies where novel cytoskeletal proteins such as BILBO1 has been identified. BILBO1 is required for the biogenesis of important cytoskeletal structures and parasite survival. Thus, BILBO1 represents an important target for intervention in pathogenic trypanosomes.An image showing immunoflourescence labeling of a bloodstream culture form, trypanosoma brucei cell probed with an anti-BILBO1 antibody is shown in this figure. Additionally, glucose and threonine metabolism have been described using DEAE cellulose purified trypanosomes and the enzymes involved in these processes have been experimentally validated. A schematic representation of glucose and threonine metabolism in the bloodstream-formed trypanosomes is shown in this figure.Purification of African trypanosomes from blood by anion exchanges DEAE cellulose columns with recent improvements remains the gold standard for trypanosome detection, not only in natural hosts with low parasitemias in endemic areas, but also for the requirement of parasites in large numbers for experimental investigations. Investigations have allowed the identification of trypanosome molecules playing essential roles in parasite survival and growth. Identified targets might represent the basis for a future vaccine with potential antigens for both humans and animals in a one health approach.

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Research

JoVE Journal

Immunology and Infection

Depletion of Specific Cell Populations by Complement Depletion

The purification of immune cell populations is often required in order to study their unique functions. In particular, molecular approaches such as real-time PCR and microarray analysis require the isolation of cell populations with high purity. Commonly used purification strategies include fluorescent activated cell sorting (FACS), magnetic bead separation and complement depletion. Of the three strategies, complement depletion offers the advantages of being fast, inexpensive, gentle on the cells and a high cell yield. The complement system is composed of a large number of plasma proteins that when activated initiate a proteolytic cascade culminating in the formation of a membrane-attack complex that forms a pore on a cell surface resulting in cell death1. The classical pathway is activated by IgM and IgG antibodies and was first described as a mechanism for killing bacteria. With the generation of monoclonal antibodies (mAb), the complement cascade can be used to lyse any cell population in an antigen-specific manner. Depletion of cells by the complement cascade is achieved by the addition of complement fixing antigen-specific antibodies and rabbit complement to the starting cell population. The cells are incubated for one hour at 37&deg;C and the lysed cells are subsequently removed by two rounds of washing. MAb with a high efficiency for complement fixation typically deplete 95-100% of the targeted cell population. Depending on the purification strategy for the targeted cell population, complement depletion can be used for cell purification or for the enrichment of cell populations that then can be further purified by a subsequent method.

To effectively study the function of immune cell populations their purification is often required. Complement depletion is a fast and inexpensive technique for the isolation of immune cell populations with high purity.

The purification of immune cell populations is often required in order to study their unique functions.  In particular, molecular approaches such as ...

Isolation of Mouse Peritoneal Cavity Cells

The peritoneal cavity is a membrane-bound and fluid-filled abdominal cavity of mammals, which contains the liver, spleen, most of the gastro-intestinal tract and other viscera. It harbors a number of immune cells including macrophages, B cells and T cells. The presence of a high number of na&iuml;ve macrophages in the peritoneal cavity makes it a preferred site for the collection of na&iuml;ve tissue resident macrophages (1). The peritoneal cavity is also important to the study of B cells because of the presence of a unique peritoneal cavity-resident B cell subset known as B1 cells in addition to conventional B2 cells. B1 cells are subdivided into B1a and B1b cells, which can be distinguished by the surface expression of CD11b and CD5. B1 cells are an important source of natural IgM providing early protection from a variety of pathogens (2-4). These cells are autoreactive in nature (5), but how they are controlled to prevent autoimmunity is still not understood completely. On the contrary, CD5+ B1a cells possess some regulatory properties by virtue of their IL-10 producing capacity (6). Therefore, peritoneal cavity B1 cells are an interesting cell population to study because of their diverse function and many unaddressed questions associated with their development and regulation. The isolation of peritoneal cavity resident immune cells is tricky because of the lack of a defined structure inside the peritoneal cavity. Our protocol will describe a procedure for obtaining viable immune cells from the peritoneal cavity of mice, which then can be used for phenotypic analysis by flow cytometry and for different biochemical and immunological assays.

The peritoneal cavity in mammals contains different immune cell populations crucial for innate immune responses. An efficient isolation method is required for biochemical and functional analyses of these cells. Here we provide a comprehensive method for the isolation of peritoneal cavity cells in the mouse.

The peritoneal cavity is a membrane-bound and fluid-filled abdominal cavity of mammals, which contains the liver, spleen, most of the gastro-intestinal ...

Purification of Specific Cell Population by Fluorescence Activated Cell Sorting (FACS)

Experimental and clinical studies often require highly purified cell populations.  FACS is a technique of choice to purify cell populations of known phenotype.  Other bulk methods of purification include panning, complement depletion and magnetic bead separation.  However, FACS has several advantages over other available methods.  FACS is the preferred method when very high purity of the desired population is required, when the target cell population expresses a very low level of the identifying marker or when cell populations require separation based on differential marker density.  In addition, FACS is the only available purification technique to isolate cells based on internal staining or intracellular protein expression, such as a genetically modified fluorescent protein marker.  FACS allows the purification of individual cells based on size, granularity and fluorescence.  In order to purify cells of interest, they are first stained with fluorescently-tagged monoclonal antibodies (mAb), which recognize specific surface markers on the desired cell population (1).  Negative selection of unstained cells is also possible.  FACS purification requires a flow cytometer with sorting capacity and the appropriate software.  For FACS, cells in suspension are passed as a stream in droplets with each containing a single cell in front of a laser.  The fluorescence detection system detects cells of interest based on predetermined fluorescent parameters of the cells.  The instrument applies a charge to the droplet containing a cell of interest and an electrostatic deflection system facilitates collection of the charged droplets into appropriate collection tubes (2).  The success of staining and thereby sorting depends largely on the selection of the identifying markers and the choice of mAb.  Sorting parameters can be adjusted depending on the requirement of purity and yield.  Although FACS requires specialized equipment and personnel training, it is the method of choice for isolation of highly purified cell populations.

Purification of Specific Cell Population by Fluorescence Activated Cell Sorting FACS

For many scientific studies requiring a biological and chemical analysis of cell populations the cells must be in a high state of purity. Fluorescence activated cell sorting (FACS) is a superior method in which to obtain pure cell populations.

Experimental and clinical studies often require highly purified cell populations.  FACS is a technique of choice to purify cell populations of known ...

Expansion, Purification, and Functional Assessment of Human Peripheral Blood NK Cells

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of NK cells and ability to expand in vitro has restricted development of NK cell immunotherapy. Here we describe a method to efficiently expand vast quantities of functional NK cells ex vivo using K562 cells expressing membrane-bound IL21, as an artificial antigen-presenting cell (aAPC).
NK cell adoptive therapies to date have utilized a cell product obtained by steady-state leukapheresis of the donor followed by depletion of T cells or positive selection of NK cells. The product is usually activated in IL-2 overnight and then administered the following day 1. Because of the low frequency of NK cells in peripheral blood, relatively small numbers of NK cells have been delivered in clinical trials.
The inability to propagate NK cells in vitro has been the limiting factor for generating sufficient cell numbers for optimal clinical outcome. Some expansion of NK cells (5-10 fold over 1-2 weeks) has be achieved through high-dose IL-2 alone 2. Activation of autologous T cells can mediate NK cell expansion, presumably also through release of local cytokine 3. Support with mesenchymal stroma or artificial antigen presenting cells (aAPCs) can support the expansion of NK cells from both peripheral blood and cord blood 4. Combined NKp46 and CD2 activation by antibody-coated beads is currently marketed for NK cell expansion (Miltenyi Biotec, Auburn CA), resulting in approximately 100-fold expansion in 21 days.
Clinical trials using aAPC-expanded or -activated NK cells are underway, one using leukemic cell line CTV-1 to prime and activate NK cells5 without significant expansion. A second trial utilizes EBV-LCL for NK cell expansion, achieving a mean 490-fold expansion in 21 days6. The third utilizes a K562-based aAPC transduced with 4-1BBL (CD137L) and membrane-bound IL-15 (mIL-15)7, which achieved a mean NK expansion 277-fold in 21 days. Although, the NK cells expanded using K562-41BBL-mIL15 aAPC are highly cytotoxic in vitro and in vivo compared to unexpanded NK cells, and participate in ADCC, their proliferation is limited by senescence attributed to telomere shortening8. More recently a 350-fold expansion of NK cells was reported using K562 expressing MICA, 4-1BBL and IL159.
Our method of NK cell expansion described herein produces rapid proliferation of NK cells without senescence achieving a median 21,000-fold expansion in 21 days.

Here we describe a method to efficiently expand and purify large numbers of human NK cells and assess their function.

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of ...

Purification and Visualization of Lipopolysaccharide from Gram-negative Bacteria by Hot Aqueous-phenol Extraction

Lipopolysaccharide (LPS) is a major component of Gram-negative bacterial outer membranes. It is a tripartite molecule consisting of lipid A, which is embedded in the outer membrane, a core oligosaccharide and repeating O-antigen units that extend outward from the surface of the cell1, 2. LPS is an immunodominant molecule that is important for the virulence and pathogenesis of many bacterial species, including Pseudomonas aeruginosa, Salmonella species, and Escherichia coli3-5, and differences in LPS O-antigen composition form the basis for serotyping of strains. LPS is involved in attachment to host cells at the initiation of infection and provides protection from complement-mediated killing; strains that lack LPS can be attenuated for virulence6-8. For these reasons, it is important to visualize LPS, particularly from clinical isolates. Visualizing LPS banding patterns and recognition by specific antibodies can be useful tools to identify strain lineages and to characterize various mutants. 
In this report, we describe a hot aqueous-phenol method for the isolation and purification of LPS from Gram-negative bacterial cells. This protocol allows for the extraction of LPS away from nucleic acids and proteins that can interfere with visualization of LPS that occurs with shorter, less intensive extraction methods9. LPS prepared this way can be separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and directly stained using carbohydrate/glycoprotein stains or standard silver staining methods. Many anti-sera to LPS contain antibodies that cross-react with outer membrane proteins or other antigenic targets that can hinder reactivity observed following Western immunoblot of SDS-PAGE-separated crude cell lysates. Protease treatment of crude cell lysates alone is not always an effective way of removing this background using this or other visualization methods. Further, extensive protease treatment in an attempt to remove this background can lead to poor quality LPS that is not well resolved by any of the aforementioned methods. For these reasons, we believe that the following protocol, adapted from Westpahl and Jann10, is ideal for LPS extraction.

We describe a modified hot aqueous-phenol extraction method for purifying lipopolysaccharide (LPS) from Gram-negative bacteria. Once extracted, the LPS can be subsequently analyzed by SDS-PAGE and visualized by direct staining or Western immunoblot.

Lipopolysaccharide (LPS)  is a major component of Gram-negative bacterial outer membranes. It is a tripartite molecule consisting of  lipid A, which is ...

Purification of Pathogen Vacuoles from Legionella-infected Phagocytes

The opportunistic pathogen Legionella pneumophila is an amoeba-resistant bacterium, which also replicates in alveolar macrophages thus causing the severe pneumonia "Legionnaires' disease"1. In protozoan and mammalian phagocytes, L. pneumophila employs a conserved mechanism to form a specific, replication-permissive compartment, the "Legionella-containing vacuole" (LCV). LCV formation requires the bacterial Icm/Dot type IV secretion system (T4SS), which translocates as many as 275 "effector" proteins into host cells. The effectors manipulate host proteins as well as lipids and communicate with secretory, endosomal and mitochondrial organelles2-4.
The formation of LCVs represents a complex, robust and redundant process, which is difficult to grasp in a reductionist manner. An integrative approach is required to comprehensively understand LCV formation, including a global analysis of pathogen-host factor interactions and their temporal and spatial dynamics. As a first step towards this goal, intact LCVs are purified and analyzed by proteomics and lipidomics.
The composition and formation of pathogen-containing vacuoles has been investigated by proteomic analysis using liquid chromatography or 2-D gel electrophoresis coupled to mass-spectrometry. Vacuoles isolated from either the social soil amoeba Dictyostelium discoideum or mammalian phagocytes harboured Leishmania5, Listeria6, Mycobacterium7, Rhodococcus8, Salmonella9 or Legionella spp.10. However, the purification protocols employed in these studies are time-consuming and tedious, as they require e.g. electron microscopy to analyse LCV morphology, integrity and purity. Additionally, these protocols do not exploit specific features of the pathogen vacuole for enrichment.
The method presented here overcomes these limitations by employing D. discoideum producing a fluorescent LCV marker and by targeting the bacterial effector protein SidC, which selectively anchors to the LCV membrane by binding to phosphatidylinositol 4-phosphate (PtdIns(4)P)3,11 . LCVs are enriched in a first step by immuno-magnetic separation using an affinity-purified primary antibody against SidC and a secondary antibody coupled to magnetic beads, followed in a second step by a classical Histodenz density gradient centrifugation12,13 (Fig. 1).
A proteome study of isolated LCVs from D. discoideum revealed more than 560 host cell proteins, including proteins associated with phagocytic vesicles, mitochondria, ER and Golgi, as well as several GTPases, which have not been implicated in LCV formation before13. LCVs enriched and purified with the protocol outlined here can be further analyzed by microscopy (immunofluorescence, electron microscopy), biochemical methods (Western blot) and proteomic or lipidomic approaches.

Purification of Pathogen Vacuoles from Legionella-infected Phagocytes

This article describes a method for the isolation and purification of intact Legionella-containing vacuoles (LCVs) from amoeba and macrophages. The two-step protocol comprises LCV enrichment by immuno-magnetic separation using an antibody against a bacterial LCV marker and further purification by density gradient centrifugation.

The opportunistic pathogen Legionella pneumophila is an amoeba-resistant bacterium, which also replicates in alveolar macrophages thus causing the severe ...

Antigens Protected Functional Red Blood Cells By The Membrane Grafting Of Compact Hyperbranched Polyglycerols

Red blood cell (RBC) transfusion is vital for the treatment of a number of acute and chronic medical problems such as thalassemia major and sickle cell anemia 1-3. Due to the presence of multitude of antigens on the RBC surface (~308 known antigens 4), patients in the chronic blood transfusion therapy develop alloantibodies due to the miss match of minor antigens on transfused RBCs 4, 5. Grafting of hydrophilic polymers such as polyethylene glycol (PEG) and hyperbranched polyglycerol (HPG) forms an exclusion layer on RBC membrane that prevents the interaction of antibodies with surface antigens without affecting the passage of small molecules such as oxygen ,glucose, and ions3. At present no method is available for the generation of universal red blood donor cells in part because of the daunting challenge presented by the presence of large number of antigens (protein and carbohydrate based) on the RBC surface and the development of such methods will significantly improve transfusion safety, and dramatically improve the availability and use of RBCs. In this report, the experiments that are used to develop antigen protected functional RBCs by the membrane grafting of HPG and their characterization are presented. HPGs are highly biocompatible compact polymers 6, 7, and are expected to be located within the cell glycocalyx that surrounds the lipid membrane 8, 9 and mask RBC surface antigens10, 11.

The cell membrane modification of red blood cells (RBCs) with hyperbranched polyglycerol (HPG) is presented. Modified RBCs were characterized by aqueous two phase partitioning, osmotic fragility and complement mediated lysis. The camouflage of surface proteins and antigens was evaluated using the flow cytometry and Micro Typing System (MTS) blood phenotyping cards.

Red blood cell (RBC) transfusion is vital for the treatment of a number of acute and chronic medical problems such as thalassemia major and sickle cell ...

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 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 ...

Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining the role of the gene of interest in the development, differentiation, and function of NK cells. Recent advances related to chimeric antigen receptors (CARs) in cancer immunotherapy accentuate the need for an efficient method to deliver exogenous genes to effector lymphocytes. The efficiencies of lentiviral-mediated gene transductions into primary human or mouse NK cells remain significantly low, which is a major limiting factor. Recent advances using cationic polymers, such as polybrene, show an improved gene transduction efficiency in T cells. However, these products failed to improve the transduction efficiencies of NK cells. This work shows that dextran, a branched glucan polysaccharide, significantly improves the transduction efficiency of human and mouse primary NK cells. This highly reproducible transduction methodology provides a competent tool for transducing human primary NK cells, which can vastly improve clinical gene delivery applications and thus NK cell-based cancer immunotherapy.

The goal of this study was to formulate technologies that allow for successful gene transduction in primary natural killer (NK) cells. The dextran-mediated lentiviral transduction of human or mouse primary NK cells results in higher gene expression efficiencies. This method of gene transduction will vastly improve NK cell genetic manipulation.

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining ...

Isolation, Characterization, and Purification of Macrophages from Tissues Affected by Obesity-related Inflammation

Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. Diet-induced obesity (DIO) is a valuable model in studying the role of macrophage heterogeneity; however, adequate macrophage isolations are difficult to acquire from inflamed tissues. In this protocol, we outline the isolation steps and necessary troubleshooting guidelines derived from our studies for obtaining a suitable population of tissue-resident macrophages from mice following 18 weeks of high-fat (HFD) or high-fat/high-cholesterol (HFHCD) diet intervention. This protocol focuses on three hallmark tissues studied in obesity and atherosclerosis including the liver, white adipose tissues (WAT), and the aorta. We highlight how dualistic usage of flow cytometry can achieve a new dimension of isolation and characterization of tissue-resident macrophages. A fundamental section of this protocol addresses the intricacies underlying tissue-specific enzymatic digestions and macrophage isolation, and subsequent cell-surface antibody staining for flow cytometric analysis. This protocol addresses existing complexities underlying fluorescent-activated cell sorting (FACS) and presents clarifications to these complexities so as to obtain broad range characterization from adequately sorted cell populations. Alternate enrichment methods are included for sorting cells, such as the dense liver, allowing for flexibility and time management when working with FACS. In brief, this protocol aids the researcher to evaluate macrophage heterogeneity from a multitude of inflamed tissues in a given study and provides insightful troubleshooting tips that have been successful for favorable cellular isolation and characterization of immune cells in DIO-mediated inflammation.

This protocol allows a researcher to isolate and characterize tissue-resident macrophages in various hallmark inflamed tissues extracted from diet-induced models of metabolic disorders.