This work was supported by grants from the Institute of Science and Technology of the Federal District (ICyTDF, 64/2012 to EO) and the Mexican Council for Science and Technology (Conacyt, 179895 to MS).

1. Establishment and Maintenance of E. histolytica Cultures
<ol>
	<li>Grow axenically (entirely free of all other contaminating organisms) 1 x 105 trophozoites of Entamoeba histolytica strain HMl:IMSS clone A18 in 16 x 125 mm culture tubes with Teflon liner screw caps (or 1 x 106 trophozoites in a disposable T-25 flask) and 15 ml (or 50 ml in T-25 flask) of TYI-S-33 medium (TYI broth supplemented with 3% Diamond vitamin mixture, 10% heat inactivated adult bovine serum, 0.5 IU/ml penicillin and 35 &#181;g/ml streptomycin)19 in an incubator at 37 &#176;C.</li>
	<li>Harvest trophozoites during the logarithmic growth phase usually at 48-96 hr intervals (Figure&#160;1) by chilling the culture tubes for 5-10 min in an ice-water bath to release trophozoites attached to the glass culture tube.</li>
	<li>Transfer the culture into a conical tube and invert it several times to disperse the cells. Determine cell number using a hemocytometer (Neubauer chamber), and transfer an inoculum into a culture tube containing fresh TYI-S-33 medium.
	<ol>
		<li>Use low numbers of amoebas for longer incubation periods (~3 x 105 cells for ~5 days) and a higher number for shorter periods (~1 x 106 cells for ~1 day). While counted inocula are desirable, established cultures become predictable so that estimated volumes of inocula are feasible.</li>
		<li>Titrate the amount of trophozoites to optimize cell numbers for each experiment.</li>
		<li>Maintain a parallel duplicate culture to have a back-up in case of inadvertent contamination or tube breakage.</li>
	</ol>
	</li>
	<li>Cap tubes tightly and incubate them at 37 &#176;C in a 5&#176; tilted horizontal angle.</li>
	<li>Check cultures by visual inspection regularly, since an excessive growth of the culture may contain many lysed cells.</li>
</ol>
2. Establishment and Maintenance of MDCK Culture
<ol>
	<li>Grow MDCK (Madin Darby Canine Kidney) cells type I (high resistance) in disposable T-75 flasks with 10 ml of DMEM media supplemented with penicillin (100 IU/ml), streptomycin (100 mg/ml), insulin (0.08 U/ml) and 10% neonate calf serum in a humidified atmosphere of 5% CO2 and 95% air at 37 &#176;C.</li>
	<li>To split the cells, check them on an inverted microscope. Split cells when they are ~ 85-100% confluent.</li>
	<li>Use a vacuum aspirator in a sterile hood and a sterile glass Pasteur pipette to remove media from the culture. To avoid scraping cells, turn the flask upside down and aspirate media from the bottom corner.</li>
	<li>Dispense 10 ml of prewarmed PBS (140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4) into a T-75 flask (5 ml in the case of a T-25 flask) and gently rock the flask to wash the cells. Remove PBS by vacuum aspiration. Extensive washing is required since serum inhibits trypsin.</li>
	<li>Dispense 1.5 ml of 0.05% trypsin into a T-75 flask (for T-25 flask use 0.5 ml) and gently rock the flask to cover the cell layer with trypsin.</li>
	<li>Incubate for 5-10 min in the incubator (exact time depends on trypsin activity).</li>
	<li>Check for cell detachment by holding flask against the light and look for flowing cells. Cell detachment (rounded cells) can also be checked using a microscope. Often, cells release faster with a quick, gentle slap to the side of the flask.</li>
	<li>Add 15 ml of supplemented DMEM to a T-75 flask (5 ml for a T-25 flask). Resuspend cells by pipetting up and down 4 or 5 times.</li>
	<li>To propagate cells, dilute cell suspension 1:5 with fresh supplemented DMEM. Cells number can also be determined at this point using a hemocytometer but this is usually only necessary if splitting cells into special formats for certain assays.</li>
</ol>
3. Preparation of Trophozoite Total Lysates
<ol>
	<li>Detach trophozoites from culture tubes by chilling the tube for 5-10 min in an ice-water bath. Transfer the culture aseptically into a conical tube and centrifuge at 360 x g for 10 min at 4 &#176;C. Carefully discard the supernatant by decantation, add ice-cold PBS to the pellet, invert the tube several times to disperse the cells and centrifuge again at 360 x g for 10 min. Repeat this step twice to remove all traces of TYI-S-33 medium.</li>
	<li>Determine trophozoites number using a hemocytometer.</li>
	<li>Lyse trophozoites by freeze-thaw cycles. Snap-freeze a measured inoculum of trophozoites (600,000 trophozoites/ml) diluted in ice-cold PBS, in liquid nitrogen for 1 min. Unfreeze the sample at 4 &#176;C and vortex vigorously. Repeat freezing and thawing 3 times to complete cell lysis.</li>
	<li>Activate proteases present in the lysates with 0.02% &#946;-mercaptoethanol.</li>
</ol>
4. Preparation of Trophozoite Secreted Products
<ol>
	<li>Perform steps 3.1 to 3.2.</li>
	<li>Determine cells number and viability at this point using a hemocytometer and applying a trypan blue exclusion test20:
	<ol>
		<li>Dilute 9 x 106 trophozoites in 3 ml ice-cold PBS and transfer cells into a 50 ml conical tube. Take 10 &#181;l of this suspension and add 1 &#181;l of 0.4% trypan blue stock solution pH 7.2.</li>
		<li>Use a Neubauer chamber to count cells at low magnification.</li>
		<li>Count all cells and separate the number of blue cells, since blue cells have taken up the dye and must be considered dead.</li>
		<li>Calculate the percentage of viable cells by dividing the number of viable cells by the total number of cells and multiplying by 100.</li>
	</ol>
	</li>
	<li>Transfer the conical tube containing the trophozoite suspension into the incubator at 37 &#176;C for 2 hr in a 5&#176; tilted horizontal angle.</li>
	<li>To collect the secreted products, centrifuge tubes at 360 x g for 10 min. Use a disposable and sterile syringe and carefully collect the supernatant, avoiding contamination of samples with trophozoites from the pellet. Eliminate any transferred cells by passing the supernatant through a 0.22 &#956;m cellulose acetate membrane. Activate proteases with 0.02% &#946;-mercaptoethanol.</li>
	<li>To discard putative unwanted contamination with molecules released from dead cells, reapply the trypan blue exclusion test for cell viability. Viable and total number of cells should be the same as obtained in step 4.2. If this is not the case, the collected supernatant may not only contain secreted proteins and should be discarded.</li>
</ol>
5. Interaction of MDCK Cells with Trophozoites, Trophozoite Lysates or Secreted Products
<ol>
	<li>Incubate confluent MDCK cell monolayers with live trophozoites (an MDCK-to-amoeba ratio of 1:1), trophozoite lysates (an MDCK-to-amoeba ratio of 1:2) or secreted products (an MDCK-to-amoeba ratio of 1:10) at 37 &#176;C for 2 and 30 min (Figure&#160;2A).</li>
	<li>Wash epithelial cells five times with ice-cold PBS to eliminate unbound molecules or trophozoites.</li>
</ol>
6. Preparation of Samples for Immunofluorescence
<ol>
	<li>Culture MDCK cells on sterile glass coverslips placed inside a 24-multiwell cell culture dish. Inspect the cells on the inverted microscope until they reach 100% of confluence. Then, replace medium with 1 ml of fresh supplemented DMEM medium and transfer the plate to a 37 &#176;C incubator for 24 hr more.</li>
	<li>Remove the medium using a vacuum aspirator and a sterile glass Pasteur pipette and add 1 ml of warm PBS to each well. Remove PBS and repeat the wash with fresh PBS. Take care not to scrape cells from the bottom of the well with the glass pipette.</li>
	<li>Add different conditions of trophozoites diluted in 1 ml of warm PBS: live trophozoites (T), trophozoite total lysates (TL) and secreted products (SP).
	<ol>
		<li>Put the culture plate in the incubator for 2 or 30 min.</li>
		<li>Prepare two control conditions: one named time 0 min, where MDCK cells should be not incubated with E. histolytica but only with 1 ml of warm PBS; and another condition as secondary antibodies control, in this case incubate MDCK with T, TL or SP for 2 or 30 min and omit incubation with primary antibodies.</li>
	</ol>
	</li>
	<li>Wash epithelial cells five times with cold PBS to eliminate unbound molecules or trophozoites.</li>
	<li>Fix and permeabilize the cells with 1 ml of 96% ethanol for 30 min at -20 &#176;C.</li>
	<li>To remove ethanol, perform three quick washes with 1 ml of PBS at room temperature.</li>
	<li>Perform the following steps in a humid chamber to avoid drying of samples:
	<ol>
		<li>Use any available flat box that can be closed and filled with a wet paper towel on which samples can be safely placed. For example, an empty pipette tip box serves this purpose well.</li>
		<li>Inside the chamber, evenly place a Parafilm layer and pipette 25 &#956;l of blocking solution (0.5% BSA) for each coverslip.</li>
		<li>Take coverslips out of the wells with fine forceps, carefully remove excess liquid from the edge with a filter paper and put coverslip into the drop containing blocking solution (0.5% BSA)with the cells facing the blocking solution. Incubate the samples for 1 hr at room temperature.</li>
	</ol>
	</li>
	<li>Prepare a mixture of primary antibodies in PBS: IgM mouse anti-EhCPADH112 (m&#945;-EhCPADH112; 1:10 dilution) and IgG rabbit anti-ZO-1 (p&#945;-ZO-1; 1:100 dilution).</li>
	<li>Place a fresh sheet of Parafilm in the humid box and pipette 25 &#956;l of antibodies solution for each coverslip.</li>
	<li>Lift the coverslips from the blocking solution, dry any excess liquid at the edges using a filter paper, and place them into the drops with the cells facing the antibody solution. Incubate the samples overnight at 4 &#176;C.</li>
	<li>Put each coverslip into a well of a 24-multiwell (cell side on top) and add 1 ml of PBS.
	<ol>
		<li>Wash five times with 1 ml of PBS to remove unbound molecules.</li>
	</ol>
	</li>
	<li>Prepare a mixture of fluorescent secondary antibodies in PBS: goat anti-IgM mouse coupled to FITC (1:100 dilution) and goat anti-IgG rabbit coupled to TRITC (1:50 dilution).</li>
	<li>Place a fresh sheet of Parafilm into the humid box and pipette 25 &#181;l of the secondary antibodies solution for each coverslip.</li>
	<li>Transfer the samples as in 6.10 and incubate for 1 hr at room temperature in the dark to avoid bleaching of the fluorescent dyes. Repeat step 6.11.1.</li>
	<li>For nuclear staining, incubate for 3 min with 200 &#956;l of 0.05 mM DAPI solution at room temperature and protect from light. Repeat step 6.11.1.</li>
	<li>To conserve the fluorescent dyes, place the coverslips (cells facing down) into 5 &#956;l Vecta Shield antifade mounting solution on glass microscope slides. Seal the cover slips using nail polish to avoid sample drying.</li>
	<li>For long term storage, keep slides in a microscope slide box at -20 &#176;C.</li>
	<li>Analyze preparations by confocal microscopy through Z-stack sections and xz-planes (Figure&#160;2A).</li>
</ol>
7. Incubation of Trophozoites with Protease Inhibitors or Specific Antibody
<ol>
	<li>Repeat steps 3.1 to 3.2. For each experimental condition, incubate 1 x 105 trophozoites (resuspended in 50 &#956;l PBS) with protease inhibitors (1 mM Complete and 40 &#181;g/ml E-64) or 30 &#181;g monoclonal antibody against EhCPADH112 (m&#945;EhCPADH112)21 for 20 min at 4 &#176;C (Figure&#160;2B). Use these preparations for co-incubation assays as described in step 8.5.</li>
</ol>
8. Measurement of Transepithelial Electrical Resistance (TER)
<ol>
	<li>Culture MDCK cells on sterile transwell permeable supports (0.4 &#956;m pore size). In the upper compartment, place 100 &#956;l of cellular suspension and in the lower compartment place 600 &#956;l of supplemented DMEM medium.
	<ol>
		<li>Check the medium level periodically. Fresh medium can be added as required.</li>
		<li>Inspect the cells on an inverted microscope until they reach 100% of confluence. Change medium every 2-3 days.</li>
		<li>To improve cell attachment (if necessary), equilibrate the transwell filters overnight at 37 &#176;C in DMEM before adding the cell suspension.</li>
	</ol>
	</li>
	<li>Sterilize STX2 electrodes in the hood by immersion in ethanol and then in sterile PBS for 15 min each, under UV light. Connect electrodes to an EVOM epithelial voltohmmeter and select resistance mode.</li>
	<li>Collect medium from the upper compartment of each transwell and pool them. Take 50 &#956;l of this media mixture and combine it with 50 &#956;l of trophozoites suspension (1 x 105 trophozoites in PBS) or with only PBS (control). Use a similar mixture for each transwell.</li>
	<li>Add mixtures to the upper chamber of each transwell containing the MDCK cells. Experimental conditions include MDCK cells without trophozoites (control), a co-culture with trophozoites or with trophozoites pre-incubated with protease inhibitors or m&#945;EhCPADH112. To eliminate the resistance provided by filters, use transwells incubated with medium only. For each experimental condition use at least three transwells.</li>
	<li>Immediately, measure TER by dipping the STX2 electrodes into each transwell.
	<ol>
		<li>Ensure that the longer electrode touches the bottom of the lower chamber and that the shorter electrode is covered by medium in the upper compartment (see Figure&#160;2B).</li>
		<li>Make sure to hold the electrodes perpendicularly each time. This will improve reproducibility, significantly. Avoid moving or tilting the electrode during the measurements as this will lead to variation of data.</li>
	</ol>
	</li>
	<li>This measurement should be considered the initial TER value. Then, monitor the TER during the following 30 min.</li>
	<li>To calculate the final TER value, subtract the TER value of filters only. To compare results from different experiments normalize each data point to the initial values, taking these as 100% (Figure&#160;2B).</li>
</ol>

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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+of+imaging+techniques+to+studies+of+epithelial+tight+junctions.">Applications of imaging techniques to studies of epithelial tight junctions.</a> Adv Drug Deliv Rev. 57, 111-121 (2005).</li></ol>

The authors have nothing to disclose.

In order to study in vitro host-pathogen interactions during epithelial infection by E. histolytica, it is crucial to work with well-established cultures of both epithelial cells and trophozoites. For example, formerly, E. histolytica cultures had usually been established in association with certain species of bacteria or trypanosomatids22,23. However, co-cultivation of E. histolytica cultures is counterproductive for the study of host-pathogen interactions because observed ...

Entamoeba histolytica is a single cell protozoan responsible of human amoebiasis, an intestinal infection causing inflammation and diarrhea. E. histolytica infects up to 50 million individuals yearly, but only about 10% of infected people develop the symptoms associated to amoebiasis1. Infection occurs upon ingestion of contaminated food or water containing E. histolytica cysts. In the intestine, cysts produce live trophozoites that adhere to colon mucin and proliferate2. Trophozoites usually form cysts that are excreted via stools. In other cases and for yet unknown reasons, trophozoites break the intestinal epithelial layer and invade underlying tissues. In worst cases, they enter the blood stream and affect other organs such as the liver3.
Breaking the epithelial barrier requires disruption of epithelial transmembranal structures that maintain cells joined. Epithelial cell contacts are formed by the apical junctional complex consisting of tight (TJ) and adherens junctions (AJ), and desmosomes4. The most apical junctions are TJ, and therefore, they are the first barrier affronted by E. histolytica and some other pathogens during host invasion. TJ are comprised of transmembranal adhesion receptors such as claudins, occludin and junctional adhesion molecules (JAM) that engage in homo- or heterophilic interactions with receptors of the neighboring cell. They are intracellularly bound by scaffold molecules of the zonula occludens (ZO) family that connect adhesion receptors to actin cytoskeleton to provide further mechanical strength to the epithelium. TJ are responsible for sealing intestinal tissue from the gut lumen, preventing excessive water and solute leakage. Thus, after TJ are disrupted by the parasite, tissues are invaded. E. histolytica secretes several molecules such as: (i) those involved in adhesion of amoebae to target cells5; (ii) membrane-active factors participating in killing of host cells by exocytosis, for example the ion channel-forming peptides termed amoebapores6,7; and (iii) proteinases that degrade extracellular matrix proteins and mediate tissue disintegration5,8,9.
The cysteine protease EhCP112 and the adhesion molecule EhADH112 that together form the EhCPADH112 complex are two E. histolytica virulence proteins that play a major role in the disassembly of TJ 10. Live trophozoites, their total lysates and secreted products induce molecular changes in the TJ complex and functional disturbance of the epithelial barrier. In this study, it is shown that EhCP112 and EhADH112 interact with occludin and claudin-1 proteins leading to internalization and degradation of cell proteins, thus facilitating E. histolytica entrance through the paracellular pathway.
Our data and those of other groups11-17strongly suggest the necessity of specific host-pathogen interactions that allow parasite invasion. Unraveling the molecular basis of these interactions is of utmost importance for a better understanding of amoebiasis pathogenesis. Selective disturbance of TJ by trophozoites, characterized by increased paracellular permeability, can be measured by a decrease in transepithelial electrical resistance (TER). The transference of parasitic proteins towards host epithelia can be determined by immunofluorescence staining and confocal laser microscopy, a method that can also reveal co-localization of amoeba virulence factors with epithelial junctional markers indicating possible direct interactions. In this article, we describe in detail how epithelial cells and trophozoites are cultivated, harvested and manipulated to examine host-pathogen interactions and their consequences.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Entamoeba histolytica HM1:IMSS, Clone A&#160;</td>
			<td>IMSS Hospital, Mexico</td>
			<td>Without/number</td>
			<td>Virulent trophozoites18&#160;</td>
		</tr>
		<tr>
			<td rowspan="8">TYI broth</td>
			<td rowspan="8">Becton, Dickinson and Company 
			Merck 
			Merck 
			Merck 
			J.T. Baker 
			Reproquifin 
			SIGMA-Aldrich 
			SIGMA-Aldrich</td>
			<td rowspan="8">211862 
			K35625437 626 
			21578 
			4873 
			3252-01 
			CAS 50-81-7 
			C7880 
			F-5879</td>
			<td rowspan="8">3.45% BBL Biosate peptone 
			58 mM glucose 
			39 mM NaCl 
			5 mM KH2PO4 
			6.5 mM K2HPO4 
			16.3 mM ascorbic acid 
			8.1 mM L-cysteine 
			&#160;0.1 mM ferric ammonium citrate, adjust pH 6.819</td>
		</tr>
		<tr>
			<td>Bovine serum adult</td>
			<td>Microlab , Labs., Mex.</td>
			<td>SU146</td>
			<td>Use at 10% and inactivated to 56&#176; C for 30 min</td>
		</tr>
		<tr>
			<td>Diamond&#160; vitamin&#160; mixture- Tween 80</td>
			<td>In vitro</td>
			<td>SR-07</td>
			<td>Use at 3%</td>
		</tr>
		<tr>
			<td>Penicillin&#160;</td>
			<td>Lakeside,&#160; M&#233;x.</td>
			<td>34564SSA IV</td>
			<td>0.5 IU/mL</td>
		</tr>
		<tr>
			<td>Streptomycin&#160;</td>
			<td>Lakeside,&#160; M&#233;x.</td>
			<td>75757SSA IV</td>
			<td>35 &#181;g/ mL</td>
		</tr>
		<tr>
			<td>Pyrex 15 mL screw cap culture tubes with PTFE lined phenolic caps</td>
			<td>Corning-Pyrex</td>
			<td>9826-16X</td>
			<td>16x125 mm, capacity 15 mL and caps fabricated from special formula resistant to effects of temperature</td>
		</tr>
		<tr>
			<td>Cell culture plates, 6 Well</td>
			<td>Corning-Costar</td>
			<td>3516</td>
			<td>Sterile plates, well diameter 34.8 mm and growth area 9.5 cm2.&#160; Rings on lid prevent cross-contamination</td>
		</tr>
		<tr>
			<td>25cm2 cell culture flask</td>
			<td>Corning-Costar</td>
			<td>430168</td>
			<td>Canted neck flasks</td>
		</tr>
		<tr>
			<td>MDCK (Madin Darby canine kidney) type I</td>
			<td>American Type Culture Collection</td>
			<td>CCL34</td>
			<td>Kidney epithelial cells grown between the 60th and 90th passage</td>
		</tr>
		<tr>
			<td>DMEM medium&#160;</td>
			<td>Gibco&#160;</td>
			<td>12800-017</td>
			<td>Dulbecco&#39;s Modified Eagle Medium with high glucose.</td>
		</tr>
		<tr>
			<td>Neonate Calf Serum</td>
			<td>In vitro</td>
			<td>S-02</td>
			<td>Use at 10%.&#160;&#160;</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin mixture&#160;</td>
			<td>In vitro&#160;</td>
			<td>A-01</td>
			<td>Stock solution 10,000 U/&#181;g/mL</td>
		</tr>
		<tr>
			<td>Insulin&#160;&#160;</td>
			<td>AMSA</td>
			<td>398MJ94SSA IV</td>
			<td>Stock solution 100 IU/mL</td>
		</tr>
		<tr>
			<td>Trypsin solution&#160;</td>
			<td>In vitro</td>
			<td>EN-005</td>
			<td>0.05% enzyme solution without calcium and magnesium</td>
		</tr>
		<tr>
			<td>75cm2 cell culture flask</td>
			<td>Corning-Costar</td>
			<td>430720</td>
			<td>Canted neck flasks for trophozoite culture in TYI-S-33 medium</td>
		</tr>
		<tr>
			<td>Transwell permeable supports</td>
			<td>Corning-Costar</td>
			<td>3470</td>
			<td>0.4. &#181;m polyster membrane, 6.5 mm insert in 24 well plate, growth area 0.3 cm2</td>
		</tr>
		<tr>
			<td>24 well cell culture dish&#160;&#160;</td>
			<td>Corning-Costar</td>
			<td>3524</td>
			<td>Clear polystyrene, treated for optimal cell attachment, sterilized by gamma radiation and certified non-pyrogenic</td>
		</tr>
		<tr>
			<td>Complete Mini</td>
			<td>Roche</td>
			<td>11836 153 001</td>
			<td>Protease inhibitor cocktail inhibits a broad spectrum of serine, cysteine and metallo-proteases. Final concentration 1 mM&#160;</td>
		</tr>
		<tr>
			<td>Trans-epoxysuccinyl-L-leucylamido (4-guanidino) butane (E-64)</td>
			<td>SIGMA-Aldrich</td>
			<td>E3132</td>
			<td>Cystein protease inhibitor, final concentration 40 &#181;g/mL</td>
		</tr>
		<tr>
			<td>p&#945;ZO-1&#160;</td>
			<td>Invitrogen</td>
			<td>402200</td>
			<td>IgG rabbit policlonal&#160; antibody&#160; against&#160; a synthetic peptide in the middle region of the ZO-1 human protein</td>
		</tr>
		<tr>
			<td>m&#945;EhCPADH112</td>
			<td>Homemade antibody</td>
			<td>Without/ Number</td>
			<td>IgM mouse monoclonal antibody&#160; against&#160; 444-601 epitope located at C-terminal of EhCPADH11221,27</td>
		</tr>
		<tr>
			<td>FITC-goat anti-mouse IgM</td>
			<td>Zymed</td>
			<td>62-6811</td>
			<td>Fluorescein isotiocyanate (FITC)-labelled goat anti-mouse secondary&#160; antibody</td>
		</tr>
		<tr>
			<td>TRITC- goat anti-rabbit IgG (H+L)</td>
			<td>Zymed</td>
			<td>816114</td>
			<td>Tetramethyl-rhodamine isothiocyanate (TRITC)-labelled&#160; goat anti-rabbit IgG&#160; secondary antibody.</td>
		</tr>
		<tr>
			<td>STX2 Electrode</td>
			<td>World Precision Instrument&#160;</td>
			<td>102711</td>
			<td>Consists of a fixed pair of double electrodes, 4 mm wide and 1 mm thick. Each stick of the electrode pair contains a silver/silver-chloride pellet for measuring voltage and a silver electrode for passing current. For use with EVOM</td>
		</tr>
		<tr>
			<td>EVOM epithelial voltohmmeter</td>
			<td>World Precision Instrument&#160;</td>
			<td>12111</td>
			<td>Use in resistance mode and maintain unplugged during TER measurements</td>
		</tr>
		<tr>
			<td>Neubauer chamber</td>
			<td>MEARIENFELD</td>
			<td>610610</td>
			<td>Hemocytometer&#160;</td>
		</tr>
		<tr>
			<td>Leica TCS_SP5_MO</td>
			<td>Leica</td>
			<td>Without/number</td>
			<td>Laser confocal microscopy with Leica microsystems CMS Gmbh/leica Las af Lite/BIN software</td>
		</tr>
		<tr>
			<td>Vectashield</td>
			<td>Vector Laboratories, Inc.</td>
			<td>H-1000</td>
			<td>Mounting medium for fluorescence</td>
		</tr>
		<tr>
			<td>4&#180;, 6-diamino-2-phenylindole (Dapi)</td>
			<td>SIGMA</td>
			<td>D-9542</td>
			<td>0.05 mM final concentration</td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>US Biological</td>
			<td>A-1310</td>
			<td>0.5%&#160; final concentration for blocking solution</td>
		</tr>
	</tbody>
</table>

analysis of the epithelial damage produced by entamoeba histolytica infection

Entamoeba histolytica is the causative agent of human amoebiasis, a major cause of diarrhea and hepatic abscess in tropical countries. Infection is initiated by interaction of the pathogen with intestinal epithelial cells. This interaction leads to disruption of intercellular structures such as tight junctions (TJ). TJ ensure sealing of the epithelial layer to separate host tissue from gut lumen. Recent studies provide evidence that disruption of TJ by the parasitic protein EhCPADH112 is a prerequisite for E. histolytica invasion that is accompanied by epithelial barrier dysfunction. Thus, the analysis of molecular mechanisms involved in TJ disassembly during E. histolytica invasion is of paramount importance to improve our understanding of amoebiasis pathogenesis. This article presents an easy model that allows the assessment of initial host-pathogen interactions and the parasite invasion potential. Parameters to be analyzed include transepithelial electrical resistance, interaction of EhCPADH112 with epithelial surface receptors, changes in expression and localization of epithelial junctional markers and localization of parasite molecules within epithelial cells.

For a successful E. histolytica culture, two important conditions must be fulfilled: growth in axenic conditions and harvest in logarithmic phase. Previously, cultures of E. histolytica were readily established in association with certain species of bacteria or trypanosomatids22. However, nowadays it is common to have axenic cultivation of this parasite meaning an indefinite subcultivation of amoebae in an environment free of metabolizing bacteria, fungi, protozoa, or metazoan cells. Addition...

Watch this Scientific Journal Video about Analysis of the Epithelial Damage Produced by Entamoeba histolytica Infection at JoVE.com

Analysis of the Epithelial Damage Produced by Entamoeba histolytica Infection

Human infection by Entamoeba histolytica leads to amoebiasis, a major cause of diarrhea in tropical countries. Infection is initiated by pathogen interactions with intestinal epithelial cells, provoking the opening of cell-cell contacts and consequently diarrhea, sometimes followed by liver infection. This article provides a model to assess the early host-pathogen interactions to improve our understanding of amoebiasis pathogenesis.

Analysis of the Epithelial Damage Produced by Entamoeba histolytica Infection

Entamoeba histolytica is the causative agent of human amoebiasis, a major cause of diarrhea and hepatic abscess in tropical countries. Infection is ...

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JoVE Journal

Immunology and Infection

18.6K Views.  Center for Research and Advanced Studies of the National Polytechnic Institute. Human infection by Entamoeba histolytica leads to amoebiasis, a major cause of diarrhea in tropical countries. Infection is initiated by pathogen interactions with intestinal epithelial cells, provoking the opening of cell-cell contacts and consequently diarrhea, sometimes followed by liver infection. This article provides a model to assess the early host-pathogen interactions to improve our understanding of amoebiasis pathogenesis.

Video: Analysis of the Epithelial Damage Produced by Entamoeba histolytica Infection

In vitro tRNA Methylation Assay with the Entamoeba histolytica DNA and tRNA Methyltransferase Dnmt2 (Ehmeth) Enzyme

Protozoan parasites are among the most devastating infectious agents of humans responsible for a variety of diseases including amebiasis, which is one of the three most common causes of death from parasitic disease. The agent of amebiasis is the amoeba parasite Entamoeba histolytica that exists under two stages: the infective cyst found in food or water and the invasive trophozoite living in the intestine. The clinical manifestations of amebiasis range from being asymptomatic to colitis, dysentery or liver abscesses. E. histolytica is one of the rare unicellular parasite with 5-methylcytosine (5mC) in its genome. 1, 2 It contains a single DNA methyltransferase, Ehmeth, that belongs to the Dnmt2 family. 2 A role for Dnmt2 in the control of repetitive elements has been established in E. histolytica, 3 Dictyostelium discoideum 4,5 and Drosophila. 6 Our recent work has shown that Ehmeth methylates tRNAAsp, and this finding indicates that this enzyme has a dual DNA/tRNAAsp methyltransferase activity. 7 This observation is in agreement with the dual activity that has been reported for D. discoideum and D. melanogaster. 8 The functional significance of the DNA/tRNA specificity of Dnmt2 enzymes is still unknown. To address this question, a method to determine the tRNA methyltransferase activity of Dnmt2 proteins was established. In this video, we describe a straightforward approach to prepare an adequate tRNA substrate for Dnmt2 and a method to measure its tRNA methyltransferase activity.

In vitro tRNA Methylation Assay with the Entamoeba histolytica DNA and tRNA Methyltransferase Dnmt2 Ehmeth Enzyme

This protocol describes the preparation of a synthetic tRNA substrate for the Entamoeba histolytica DNA/tRNA methyltransferase 2 (Dnmt2) homolog Ehmeth and the measure of its methyltransferase activity. This experimental approach can be used for investigating the activity of other Dnmt2 proteins.

Protozoan parasites are among the most devastating infectious agents of humans responsible for a variety of diseases including amebiasis, which is one of ...

Development of a Negative Selectable Marker for Entamoeba histolytica

Entamoeba histolytica is the causative agent of amebiasis and infects up to 10% of the world's population. The molecular techniques that have enabled the up- and down-regulation of gene expression rely on the transfection of stably maintained plasmids. While these have increased our understanding of Entamoeba virulence factors, the capacity to integrate exogenous DNA into genome, which would allow reverse genetics experiments, would be a significant advantage in the study of this parasite. The challenges presented by this organism include inability to select for homologous recombination events and difficulty to cure episomal plasmid DNA from transfected trophozoites. The later results in a high background of exogenous DNA, a major problem in the identification of trophozoites in which a bona fide genomic integration event has occurred. We report the development of a negative selection system based upon transgenic expression of a yeast cytosine deaminase and uracil phosphoribosyl transferase chimera (FCU1) and selection with prodrug 5-fluorocytosine (5-FC). The FCU1 enzyme converts non-toxic 5-FC into toxic 5-fluorouracil and 5-fluorouridine-5'-monophosphate. E. histolytica lines expressing FCU1 were found to be 30 fold more sensitive to the prodrug compared to the control strain.

Development of a Negative Selectable Marker for Entamoeba histolytica

We report development of a negative selection system in E. histolytica based upon transgenic expression of a chimeric protein (FCU1) and selection with the prodrug 5-fluorocytosine. The FCU1 protein is a fusion of yeast cytosine deaminase and uracil phosphoribosyltransferase. Expression of FCU1 resulted in increased E. histolytica sensitivity towards 5-fluorocytosine.

Entamoeba histolytica is the causative agent of amebiasis and infects up to 10% of the world's population. The molecular techniques that have enabled the ...

Application of a Mouse Ligated Peyer&#x2019;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed to them and occasionally to pathogens. The gut-associated lymphoid tissue (GALT) play a key role for induction of the mucosal immune response to these microbes1, 2. To initiate the mucosal immune response, the mucosal antigens must be transported from the gut lumen across the epithelial barrier into organized lymphoid follicles such as Peyer's patches. This antigen transcytosis is mediated by specialized epithelial M cells3, 4. M cells are atypical epithelial cells that actively phagocytose macromolecules and microbes. Unlike dendritic cells (DCs) and macrophages, which target antigens to lysosomes for degradation, M cells mainly transcytose the internalized antigens. This vigorous macromolecular transcytosis through M cells delivers antigen to the underlying organized lymphoid follicles and is believed to be essential for initiating antigen-specific mucosal immune responses. However, the molecular mechanisms promoting this antigen uptake by M cells are largely unknown. We have previously reported that glycoprotein 2 (Gp2), specifically expressed on the apical plasma membrane of M cells among enterocytes, serves as a transcytotic receptor for a subset of commensal and pathogenic enterobacteria, including Escherichia coli and Salmonella enterica serovar Typhimurium (S. Typhimurium), by recognizing FimH, a component of type I pili on the bacterial outer membrane 5. Here, we present a method for the application of a mouse Peyer's patch intestinal loop assay to evaluate bacterial uptake by M cells. This method is an improved version of the mouse intestinal loop assay previously described 6, 7. The improved points are as follows: 1. Isoflurane was used as an anesthetic agent. 2. Approximately 1 cm ligated intestinal loop including Peyer's patch was set up. 3. Bacteria taken up by M cells were fluorescently labeled by fluorescence labeling reagent or by overexpressing fluorescent protein such as green fluorescent protein (GFP). 4. M cells in the follicle-associated epithelium covering Peyer's patch were detected by whole-mount immunostainig with anti Gp2 antibody. 5. Fluorescent bacterial transcytosis by M cells were observed by confocal microscopic analysis. The mouse Peyer's patch intestinal loop assay could supply the answer what kind of commensal or pathogenic bacteria transcytosed by M cells, and may lead us to understand the molecular mechanism of how to stimulate mucosal immune system through M cells.

Application of a Mouse Ligated Peyer&amp;#8217;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

M cells in a specialized follicle-associated epithelium covering Peyer&#x2019;s patches play an important role for the mucosal immunosurveillance in gut-associated lymphoid tissue. Here we described the evaluation method for bacterial transcytosis by M cells in vivo. This method provides a method to understand M-cell function in the immune system.

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed ...

Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies

Throughout the last years, the contribution of alveolar type II epithelial cells (AECII) to various aspects of immune regulation in the lung has been increasingly recognized. AECII have been shown to participate in cytokine production in inflamed airways and to even act as antigen-presenting cells in both infection and T-cell mediated autoimmunity 1-8. Therefore, they are especially interesting also in clinical contexts such as airway hyper-reactivity to foreign and self-antigens as well as infections that directly or indirectly target AECII. However, our understanding of the detailed immunologic functions served by alveolar type II epithelial cells in the healthy lung as well as in inflammation remains fragmentary. Many studies regarding AECII function are performed using mouse or human alveolar epithelial cell lines 9-12. Working with cell lines certainly offers a range of benefits, such as the availability of large numbers of cells for extensive analyses. However, we believe the use of primary murine AECII allows a better understanding of the role of this cell type in complex processes like infection or autoimmune inflammation. Primary murine AECII can be isolated directly from animals suffering from such respiratory conditions, meaning they have been subject to all additional extrinsic factors playing a role in the analyzed setting. As an example, viable AECII can be isolated from mice intranasally infected with influenza A virus, which primarily targets these cells for replication 13. Importantly, through ex vivo infection of AECII isolated from healthy mice, studies of the cellular responses mounted upon infection can be further extended.
Our protocol for the isolation of primary murine AECII is based on enzymatic digestion of the mouse lung followed by labeling of the resulting cell suspension with antibodies specific for CD11c, CD11b, F4/80, CD19, CD45 and CD16/CD32. Granular AECII are then identified as the unlabeled and sideward scatter high (SSChigh) cell population and are separated by fluorescence activated cell sorting 3.
In comparison to alternative methods of isolating primary epithelial cells from mouse lungs, our protocol for flow cytometric isolation of AECII by negative selection yields untouched, highly viable and pure AECII in relatively short time. Additionally, and in contrast to conventional methods of isolation by panning and depletion of lymphocytes via binding of antibody-coupled magnetic beads 14, 15, flow cytometric cell-sorting allows discrimination by means of cell size and granularity. Given that instrumentation for flow cytometric cell sorting is available, the described procedure can be applied at relatively low costs. Next to standard antibodies and enzymes for lung disintegration, no additional reagents such as magnetic beads are required. The isolated cells are suitable for a wide range of functional and molecular studies, which include in vitro culture and T-cell stimulation assays as well as transcriptome, proteome or secretome analyses 3, 4.

We describe the rapid isolation of primary murine type II alveolar epithelial cells (AECII) by flow cytometric negative selection. These AECII show high viability and purity and are suitable for a wide range of functional and molecular studies regarding their role in respiratory conditions such as autoimmune or infectious diseases.

Throughout  the last years, the contribution of alveolar type II epithelial cells (AECII)  to various aspects of immune regulation in the lung has been ...

Analysis of the Solvent Accessibility of Cysteine Residues on Maize rayado fino virus Virus-like Particles Produced in Nicotiana benthamiana Plants and Cross-linking of Peptides to VLPs

Mimicking and exploiting virus properties and physicochemical and physical characteristics holds promise to provide solutions to some of the world's most pressing challenges. The sheer range and types of viruses coupled with their intriguing properties potentially give endless opportunities for applications in virus-based technologies. Viruses have the ability to self- assemble into particles with discrete shape and size, specificity of symmetry, polyvalence, and stable properties under a wide range of temperature and pH conditions. Not surprisingly, with such a remarkable range of properties, viruses are proposed for use in biomaterials 9, vaccines 14, 15, electronic materials, chemical tools, and molecular electronic containers4, 5, 10, 11, 16, 18, 12.
In order to utilize viruses in nanotechnology, they must be modified from their natural forms to impart new functions. This challenging process can be performed through several mechanisms including genetic modification of the viral genome and chemically attaching foreign or desired molecules to the virus particle reactive groups 8. The ability to modify a virus primarily depends upon the physiochemical and physical properties of the virus. In addition, the genetic or physiochemical modifications need to be performed without adversely affecting the virus native structure and virus function. Maize rayado fino virus (MRFV) coat proteins self-assemble in Escherichia coli producing stable and empty VLPs that are stabilized by protein-protein interactions and that can be used in virus-based technologies applications 8. VLPs produced in tobacco plants were examined as a scaffold on which a variety of peptides can be covalently displayed 13. Here, we describe the steps to 1) determine which of the solvent-accessible cysteines in a virus capsid are available for modification, and 2) bioconjugate peptides to the modified capsids. By using native or mutationally-inserted amino acid residues and standard coupling technologies, a wide variety of materials have been displayed on the surface of plant viruses such as, Brome mosaic virus 3, Carnation mottle virus 12, Cowpea chlorotic mottle virus 6, Tobacco mosaic virus 17, Turnip yellow mosaic virus 1, and MRFV 13.

Analysis of the Solvent Accessibility of Cysteine Residues on Maize rayado fino virus Virus-like Particles Produced in Nicotiana benthamiana Plants and Cross-linking of Peptides to VLPs

A method to analyze the solvent accessibility of the thiol group of cysteine residues of Maize rayado fino virus (MRFV)-virus-like particles (VLPs) followed by a peptide cross-linking reaction is described. The method takes advantage of the availability of several chemical groups on the surface of the VLPs that can be targets for specific reactions.

Mimicking  and exploiting virus properties and physicochemical and physical  characteristics holds promise to provide solutions to some of  the world's ...

Capsular Serotyping of Streptococcus pneumoniae by Latex Agglutination

Latex agglutination reagents are widely used in microbial diagnosis, identification and serotyping. Streptococcus pneumoniae (the pneumococcus) is a major cause of morbidity and mortality world-wide. Current vaccines target the pneumococcal capsule, and there are over 90 capsular serotypes. Serotyping pneumococcal isolates is therefore important for assessing the impact of vaccination programs and for epidemiological purposes. The World Health Organization has recommended latex agglutination as an alternative method to the &#8216;gold standard&#8217; Quellung test for serotyping pneumococci. Latex agglutination is a relatively simple, quick and inexpensive method; and is therefore suitable for resource-poor settings as well as laboratories with high-volume workloads. Latex agglutination reagents can be prepared in-house utilizing commercially-sourced antibodies that are passively attached to latex particles. This manuscript describes a method of production and quality control of latex agglutination reagents, and details a sequential testing approach which is time- and cost-effective. 
This method of production and quality control may also be suitable for other testing purposes.

Capsular Serotyping of Streptococcus pneumoniae by Latex Agglutination

Latex agglutination testing is a simple, rapid and inexpensive method for serotyping Streptococcus pneumoniae, and has also been widely applied in diagnostic microbiology. This manuscript describes the in-house production of latex agglutination reagents, quality control procedures and the application of this technique to pneumococcal serotyping.

Latex agglutination reagents are widely used in microbial diagnosis, identification and serotyping. Streptococcus pneumoniae (the pneumococcus) is a major ...

Rapid Analysis of Chromosome Aberrations in Mouse B Lymphocytes by PNA-FISH

Defective DNA repair leads to increased genomic instability, which is the root cause of mutations that lead to tumorigenesis. Analysis of the frequency and type of chromosome aberrations in different cell types allows defects in DNA repair pathways to be elucidated. Understanding mammalian DNA repair biology has been greatly helped by the production of mice with knockouts in specific genes. The goal of this protocol is to quantify genomic instability in mouse B lymphocytes. Labeling of the telomeres using PNA-FISH probes (peptide nucleic acid - fluorescent in situ hybridization) facilitates the rapid analysis of genomic instability in metaphase chromosome spreads. B cells have specific advantages relative to fibroblasts, because they have normal ploidy and a higher mitotic index. Short-term culture of B cells therefore enables precise measurement of genomic instability in a primary cell population which is likely to have fewer secondary genetic mutations than what is typically found in transformed fibroblasts or patient cell lines.

DNA repair pathways are essential for maintenance of genomic integrity and preventing mutation and cancer. The goal of this protocol is to quantify genomic instability by direct observation of chromosome aberrations in metaphase spreads from mouse B cells using fluorescent in situ hybridization (FISH) for telomeric DNA repeats.

Defective DNA repair leads to increased genomic instability, which is the root cause of mutations that lead to tumorigenesis. Analysis of the frequency ...

Use of the Soft-agar Overlay Technique to Screen for Bacterially Produced Inhibitory Compounds

The soft-agar overlay technique was originally developed over 70 years ago and has been widely used in several areas of microbiological research, including work with bacteriophages and bacteriocins, proteinaceous antibacterial agents. This approach is relatively inexpensive, with minimal resource requirements. This technique consists of spotting supernatant from a donor strain (potentially harboring a toxic compound(s)) onto a solidified soft agar overlay that is seeded with a bacterial test strain (potentially sensitive to the toxic compound(s)). We utilized this technique to screen a library of Pseudomonas syringae strains for intraspecific killing. By combining this approach with a precipitation step and targeted gene deletions, multiple toxic compounds produced by the same strain can be differentiated. The two antagonistic agents commonly recovered using this technique are bacteriophages and bacteriocins. These two agents can be differentiated using two simple additional tests. Performing a serial dilution on a supernatant containing bacteriophage will result in individual plaques becoming less in number with greater dilution, whereas serial dilution of a supernatant containing bacteriocin will result a clearing zone that becomes uniformly more turbid with greater dilution. Additionally, a bacteriophage will produce a clearing zone when spotted onto a fresh soft agar overlay seeded with the same strain, whereas a bacteriocin will not produce a clearing zone when transferred to a fresh soft agar lawn, owing to the dilution of the bacteriocin.

We describe a simple method for screening bacterial cultures for the production of compounds inhibitory towards other bacteria.

The soft-agar overlay technique was originally developed over 70 years ago and has been widely used in several areas of microbiological research, ...

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+ ...

Characterization of Human Monocyte Subsets by Whole Blood Flow Cytometry Analysis

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can have pathological significance. An ideal method for examining alterations to monocytes is whole blood flow cytometry as the minimal handling of samples by this method limits artifactual cell activation. However, many different approaches are taken to gate the monocyte subsets leading to inconsistent identification of the subsets between studies. Here we demonstrate a method using whole blood flow cytometry to identify and characterize human monocyte subsets (classical, intermediate, and non-classical). We outline how to prepare the blood samples for flow cytometry, gate the subsets (ensure contaminating cells have been removed), and determine monocyte subset expression of surface markers &#8212; in this example M1 and M2 markers. This protocol can be extended to other studies that require a standard gating method for assessing monocyte subset proportions and monocyte subset expression of other functional markers.

Here we present a protocol for characterizing monocyte subsets by whole blood flow cytometry. This includes outlining how to gate the subsets and assess their expression of surface markers and giving an example of the assessment of the expression of M1 (inflammatory) and M2 markers (anti-inflammatory).

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can ...