Name: a parasite rescue and transformation assay for antileishmanial screening against intracellular leishmania donovani amastigotes in thp1 human acute monocytic leukemia cell line
Uploaded: 2012-12-30T12:00:00
Duration: 12 min 22 s
Description: Watch this Scientific Journal Video about A Parasite Rescue and Transformation Assay for Antileishmanial Screening Against Intracellular Leishmania donovani Amastigotes in THP1 Human Acute Monocytic L

The NCNPR-USDA-ARS Scientific agreement No. 58-6408-2-0009; CDMRP grant Award # W81XWH-09-2-0093 by U.S. Army Medical research and Materiel Command.

1. Maintain and Subculture THP1 Cell Culture
<ol>
 <li>Maintain THP1 cells in RPMI-1640 medium (with 10% FBS and pH 7.4) at 37 &deg;C in a 5% CO2 incubator.</li>
 <li>Subculture cells twice a week to prevent cell count from exceeding 1x106 cells/ml. This is important to maintain their transformation ability.</li>
</ol>
2. Maintain and Subculture Leishmania donovani Promastigotes Culture
<ol>
 <li>Maintain the L. donovani promastigotes (S1, sudan strain) in RPMI-1640 medium (without Sodium Bicarbonate and Sodium Pyruvate) with 10% FBS at 26 &deg;C.</li>
 <li>Subculture L. donovani promastigotes twice a week, with highest cells concentration in the range of 20-25x106 promastigotes/ml.Caution: All the media and solutions should be brought to the room temperature before use. </li>
</ol>
3. Seeding and Differentiation of the THP1 cells in a 96-well Microplate and 16-chamber Glass Culture Slide.
<ol>
 <li>Prepare a diluted THP1 culture with cell count of 2.5x105 cells/ml from a four-day-old cell culture (cell count should not exceed 106 cells/ml) in RPMI-1640 with 10% heat-inactivated FBS. Prepared 20 ml of culture for each 96-well plate and 4 ml of culture for each 16-well chamber slide.</li>
 <li>Add phorbol 12-myristate 13-acetate (PMA) (for differentiation of THP1) to diluted cell culture suspension (10 &mu;l/20 ml culture from the stock of 50 &mu;g/ml in DMSO) (final PMA concentration in diluted cells culture should be 25 ng/ml).</li>
 <li>To compare the Digital-Image-Analysis-Direct-Counting-Assay and Parasite-Rescue-Transformation- Assay, set up the assays simultaneously in clear, flat-bottom, 96-well plate and 16-chamber, glass, microscopic culture slide.</li>
 <li>Dispense 200 &mu;l of THP1-PMA-treated cells to each well or chamber.</li>
 <li>Incubate the 96-well plates and 16-well chamber slides in a 37 &deg;C, 5% CO2 incubator overnight to allow almost complete differentiation of the cells.</li>
</ol>
Note: The THP1 cells, which normally grow in suspension, are differentiated into adherent macrophages.
4. Infection of the Transformed THP1 Cells with Leishmania donovani Promastigotes 
<ol>
 <li>For infection of differentiated THP1 cell culture with Leishmania donovani promastigotes, the majority of parasites should be in the infective metacyclic stage (long cylindrical forms, ~ 5-6 day old culture).</li>
 <li>A 1:10 THP1 cell to parasite ratio is optimal for the infection in both the Digital-Image-Analysis-Direct-Counting-Assay and the Promastigote-Rescue-Transformation- Assay.</li>
 <li>Prepare a diluted culture of L. donovani promastigotes with a parasite count of 2.5x106 parasite/ml (for THP1 cells:parasites ratio = 1:10) from a 5 to 6-day-old culture in RPMI-1640 medium with 2% FBS.</li>
 <li>From the step 3.5 (after overnight differentiation of THP1 cell culture) take out the plates and chamber slides, remove the medium and wash the cell cultures once with serum-free RPMI-1640 medium.</li>
 <li>After careful washing of PMA-treated THP1 cells with serum-free, warm RPMI-1640 (~37 &deg;C) medium, replace the serum-free medium with 200 &mu;l of the diluted culture of L. donovani promastigotes (2.5x106 parasites/ml) from step 4.3. Set up the control wells of THP1 cells without the parasite and the parasites without THP1 cells in each plate and 16-well chamber slides.</li>
 <li>After adding parasite to the THP1 cell culture , incubate the plate and slides at 37 &deg;C, 5% CO2 for 24 hr to allow the parasites to infect the differentiated THP1 cells.</li>
</ol>
5. Treatment of Infected Macrophages with Test Drugs/Compounds
<ol>
 <li>Test Amphotericin B, Pentamidine and Miltefosine as standard anti-leishmanial drugs for screening. Prepare stock solutions of the drugs/test compounds in water or DMSO as mentioned in reagent table. Test each drug compound at 6 concentrations.</li>
 <li>Serially dilute (1:5) the standard drugs and test compounds in a fresh 96-well plate or 2 ml tubes (for chamber slides) with RPMI-1640 medium with 2% FBS. The drugs/test compound concentrations in this plate are 2X of final concentrations.</li>
 <li>Wash the culture plates and chamber slides infected with L. donovani promastigotes (from step 4.5) at least 5 times with serum-free, RPMI-1640 medium. Add 100 &mu;l culture medium (RPMI-1640 with 2% FBS) into each well/chamber. The multiple washings are necessary to ensure complete removal of non-internalized promastigotes.</li>
 <li>Add 100 &mu;l medium from serially-diluted standard anti-leishmanial drugs or the test compounds to each well or chamber. Set up the infected THP1 cells controls without drugs simultaneously in each plate/chamber slide.</li>
 <li>Incubate the plates and chamber slides at 37 &deg;C, 5% CO2 for 48 hr.</li>
</ol>
6. Chamber Slide Staining, Fluorescent Microscope Imaging and Image Analysis for Quantification of Infection and Effect of Drug Treatment
<ol>
 <li>After a 48 hr incubation, wash the chamber slide 3 times with serum-free RPMI-1640 medium. Peel off the plastic chambers from the slides and fix the cells by immersing the slides in methanol for 30 sec. Leave the slides in bio-hood under airflow for drying.</li>
 <li>Prepare a SYBR Green I staining solution (5X) by diluting (1:2,000) the stock (10,000X) with water.</li>
 <li>Stain the slides under dark conditions with the diluted SYBR Green I stain solution for 15 min at room temperature, wash once with water and leave the slides under the air flow for drying.</li>
 <li>Place a full glass coverslip over the stained slide with the help of mounting medium.</li>
 <li>Capture the digital images of THP1 cells: without infection (blank), with infection (control), infected cells treated with different standard drugs or test compounds in different dilutions (Figure 3, 5 and 6)) using a Nikon Eclipse 90i fluorescent microscope accompanied by NIS element AR 3.2 software.</li>
 <li>Count the macrophage nuclei (big) and the parasite nuclei (small with kinetoplast) using ImageJ (Figure 7), which is a publically-available, Java-based image processing program developed at the National Institutes of Health (<a href="http://rsb.info.nih.gov/ij/download.html" target="_blank">http://rsb.info.nih.gov/ij/download.html</a>). Express the data as the number of amastigotes per 100 transformed THP1 cells.</li>
</ol>
7. The Parasite-Rescue-Transformation-Assay: Controlled lysis of L. donovani Amastigotes-infected Macrophages
<ol>
 <li>Wash the 96-well microplate from step 5.5 three times with serum-free RPMI-1640 culture medium.</li>
 <li>Among different detergents tested at different concentrations, 0.05% SDS treatment for 30 sec is optimal for controlled lysis (maximum cell lysis with minimum loss of rescued parasites' viability).</li>
 <li>Remove the serum-free medium from each well after the last washing and add 20 &mu;l of RPMI-1640 (with 0.05% SDS) to each well. Shake the plate for 30 sec and add 180 &mu;l complete RPMI-1640 (with 10% FBS) to each well.</li>
 <li>Incubate the plates at 26 &deg;C for 48 hr for transformation of rescued amastigotes to promastigotes.</li>
</ol>
8. Quantitative Analysis of Transformed Promastigote (alamarBlue assay) 
<ol>
 <li>After a 48 hr incubation at 26 &deg;C, all the rescued live amastigotes are transformed into promastigotes (Figure 1D). Add 10 &mu;l of alamarBlue into each well of the 96-well plates.</li>
 <li>Incubate the plates at 26 &deg;C overnight.</li>
 <li>After overnight incubation, read the plates for standard fluorescence on a Fluostar Galaxy fluorimeter (BMG Lab Technologies) at 544 nm excitation, 590nm emission.</li>
 <li>Prepare the dose response curves (percent growth vs. concentration of the drug or test compound) with ExcelFit (Figures 7-9) and compute IC50/IC90 values from these curves (Table 1).</li>
</ol>
9. Standardization of THP1 Cells to Parasites Ratio 
<ol>
 <li>Standardize the THP1 cells to parasites ratio to determine sensitivity of the assay. Note: Low/high parasites numbers/infection may compromise with sensitivity and selectivity of the screening.</li>
 <li>Standardize the THP1 cells to parasites ratio by following the protocol described above for THP1 cells seeding and Leishmania promastigote infection (section 3 &amp; 4) except use different ratios of THP1 cells to parasites as 1:1.25, 1:2.5, 1:5 and 1:10.</li>
 <li>Set up the experiment both in 16-chamber slide (for image analysis) and 96-well plate (for parasite-rescue assay) formats to compare the parasite rescue and image analysis assays.</li>
</ol>
10. Standardization of different detergents for controlled cell lysis
<ol>
 <li>The primary objective of this experiment is to optimize the protocol for controlled lysis of the THP1 cells to achieve maximum/complete THP1 cells lysis without significantly affecting the viability of the rescued amastigote parasites.</li>
 <li>Test different detergents such as Tween 20, Tween 80, Triton X-100, NP-40 and SDS at different concentrations and different durations of treatment (Figure 2).</li>
 <li>THP1 cells to parasites ratio is 1:10 and other conditions are similar as above sections 1-8.</li>
 <li>Test the 0.05% SDS treatment further for different treatment times to further optimize the controlled cell lysis.</li>
</ol>

<ol>
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No conflicts of interest declared.

There are several methods available for anti-leishmanial drug screening based on macrophage-amastigote models. Assays can be done with the macrophages collected from host animals namely peritoneal exudate cells (PEC), peripheral blood monocyte cells (PBMC) 6 or bone marrow-derived macrophages (BMM) or in monocytic cell lines such as mouse (J774 and RAW264.7)7 and human (THP1, U937, HL-60)8 monocytic cells. The assays, which use dividing host cells, must ensure that the confounding effects of drug activity on both parasite and host cells number are considered. The differentiated primary macrophages collected from various sources such as mice and rats are non-dividing in nature, but these cell preparations may not have homogeneous cell populations. Monocytic cells-derived cell lines are homogenous in nature and are a better model for the macrophage-amastigote-based screening. Out of different monocytic cell lines, differentiated THP1 cells (human acute monocytic leukemia cell line) can form a non-dividing monolayer and offer an attractive alternative to primary isolated macrophages.
The macrophage-amastigote-based screening can be done in several ways. Classical microscopic evaluation based on direct cell and parasite counting9 is labor intensive. The absence of automation limits the utility of this assay. Counting of cells is time consuming and may give inaccurate determination of IC50 values since determination of parasite viability through a staining procedure is difficult. Many fluorescent dyes and monoclonal antibodies may be employed for flow cytometric assays10, 11, but these assays are also limited due to less sensitivity and limitation of time interval of drug-treatment to only one day. There are several reporter gene assays available for quantifying the growth of intracellular amastigotes12,13,14. An automated screening may be possible using reporter genes, but these assays also have certain drawbacks. First, majority of these assays require drug selection for maintaining the episomal expression of the reporter genes, which may not be ideal for a drug screening experiment. The way by which the reporter gene is introduced could also influence the physiological properties of the parasite and have an impact on the screening. If the reporter gene is the part of an episomal plasmid, the relative output of reporter may depend on the copy number of the transfected plasmid (which varies from cell to cell) rather than on the activity of the drug 14. Some reporter parasites which are transformed parasites do not need selective pressure to maintain the reporter gene; however, there could be biological consequences either by disrupting the genomic architecture or just by the presence of the foreign reporter proteins 15. In some reporter gene based assays, there are issues of sensitivity and background activity 16. Most importantly, many of the reporter gene expression assays, specially the one with GFP reporter gene15, may not differentiate between the live and dead intracellular amastigotes. Assays based on luciferase reporter gene may discern between living and dead intracellular amastigotes, but substrate and cell lysis buffer for these assays are expensive for large-scale screening17. To overcome these demerits and limitations of previous macrophage-amastigote-based screening assays, we have developed and optimized this parasite-rescue and transformation assay. This assay is based on THP1 cells, which have good homogeneity and are non-dividing in nature, as host cells.
The Parasite-Rescue-Transformation-Assay assay described here is comparable to the assay based on Digital-Image-Analysis-Direct-Counting of the intracellular amastigotes. Fluorescent and DIC microscopy, digital image analyses by ImageJ for differential counting of the macrophage nuclei and the parasite nuclei have further refined the microscopic counting assay. Capturing the images under fluorescent light filters and differential interference contrast (DIC) filters have improved the quality of the digital image for more accurate counting of the intracellular parasites. Both fluorescent and DIC images can be merged to obtain the digital images with clear macrophage cell outlines and fluorescent intracellular nuclei. The macrophage nuclei and the parasite nuclei can be differentially recognized with ImageJ. Therefore, both Digital-Image-Analysis-Direct-Counting-Assay and Parasite-Rescue-Transformation-Assay have the potential for automation and application to large-scale screening. The critical steps in the Parasite-Rescue-Transformation-Assay are: (a) repeated washings of THP1 cell cultures after exposure to Leishmania promastigotes, to ensure almost complete removal of the non-internalized promastigotes and (b) controlled lysis of the infected THP1 cells with SDS. Both the steps may also be controlled with automation and should not compromise with throughput of the assay. The second step of washings, after exposure of the Leishmania infected THP1 cells to the test drugs/compounds removes the remaining non-internalized parasites, if any. The Parasite-Rescue-Transformation-Assay offers significant advantages over existing microscopic, reporter gene and image analysis assays. The assay is simple, robust, and reproducible, can be automated for large-scale screening and therefore should have important application in screening of large compounds libraries for new anti-leishmanial drug discovery. Further, the assay can also be applied for evaluating infectivity of clinical, as well as, laboratory isolates of Leishmania in vitro.

<table border="1">
	<tbody>
		<tr>
			<td>Name of the Reagent</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Phorbol 12-Myristate 13-Acetate (PMA) </td>
			<td>Sigma-Aldrich USA</td>
			<td>P1585</td>
			<td>Required for differentiation of THP1 cells.</td>
		</tr>
		<tr>
			<td>RPMI-1640 Medium</td>
			<td>Invitrogen</td>
			<td>23400021</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Lab-TeK Chamber Slide System (16 Chamber)</td>
			<td>Thermo Scientific Nunc</td>
			<td>178599</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Clear, Flat-Bottom, 96-Well Plates</td>
			<td>BD Falcon</td>
			<td>353075</td>
			<td>Plates to perform 96-well plate assay</td>
		</tr>
		<tr>
			<td>Amphotericin B</td>
			<td>Sigma-Aldrich USA</td>
			<td>A4888</td>
			<td>Standard anti-leishmanial drug dissolve in DMSO (2 mg/ml)</td>
		</tr>
		<tr>
			<td>Pentamidine Isothionate Salt</td>
			<td>Sigma-Aldrich USA</td>
			<td>P 0547</td>
			<td>Standard anti-leishmanial drug dissolve in DMSO (2 mg/ml)</td>
		</tr>
		<tr>
			<td>Miltefosine</td>
			<td>EMD Biosciences USA</td>
			<td>475841</td>
			<td>Standard anti-leishmanial drug dissolve in serum-free RPMI-1640 medium (prepare fresh) (2 mg/ml)</td>
		</tr>
		<tr>
			<td>Sodium Stibogluconate</td>
			<td>EMD Biosciences USA</td>
			<td>567565</td>
			<td>Standard anti-leishmanial drug prepare in RPMI-1640 with 4% FBS. (1 mg/ml)</td>
		</tr>
		<tr>
			<td>Sodium Dodecyl Sulfate</td>
			<td>Sigma-Aldrich USA</td>
			<td>L 5750</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Fluorescence Microscope</td>
			<td>NIKON</td>
			<td>ECLIPSE 90i</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Fluorescence Microplate Reader</td>
			<td>BMG</td>
			<td>Polar Star Galaxy</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Mounting Medium</td>
			<td>Sigma-Aldrich USA</td>
			<td>M 1289</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>alamarBlue</td>
			<td>AbD Serotec</td>
			<td>BUF012 B</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>SYBR Green I</td>
			<td>Sigma-Aldrich USA</td>
			<td>S 9430</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate</td>
			<td>Fisher Sci.</td>
			<td>523500</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sodium Pyruvate</td>
			<td>Sigma-Aldrich USA</td>
			<td>P2256</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Sigma-Aldrich USA</td>
			<td>M6250</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Atlanta Biologicals</td>
			<td>S11050H</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Sigma-Aldrich USA</td>
			<td>P9416</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Tween 80</td>
			<td>Sigma-Aldrich USA</td>
			<td>P6474</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich USA</td>
			<td>T8787</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>NP-40</td>
			<td>Calbiochem</td>
			<td>492016</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

a parasite rescue and transformation assay for antileishmanial screening against intracellular leishmania donovani amastigotes in thp1 human acute monocytic leukemia cell line

Leishmaniasis is one of the world's most neglected diseases, largely affecting the poorest of the poor, mainly in developing countries. Over 350 million people are considered at risk of contracting leishmaniasis, and approximately 2 million new cases occur yearly1. Leishmania donovani is the causative agent for visceral leishmaniasis (VL), the most fatal form of the disease. The choice of drugs available to treat leishmaniasis is limited 2;current treatments provide limited efficacy and many are toxic at therapeutic doses. In addition, most of the first line treatment drugs have already lost their utility due to increasing multiple drug resistance 3. The current pipeline of anti-leishmanial drugs is also severely depleted. Sustained efforts are needed to enrich a new anti-leishmanial drug discovery pipeline, and this endeavor relies on the availability of suitable in vitro screening models.
In vitro promastigotes 4 and axenic amastigotes assays5 are primarily used for anti-leishmanial drug screening however, may not be appropriate due to significant cellular, physiological, biochemical and molecular differences in comparison to intracellular amastigotes. Assays with macrophage-amastigotes models are considered closest to the pathophysiological conditions of leishmaniasis, and are therefore the most appropriate for in vitro screening. Differentiated, non-dividing human acute monocytic leukemia cells (THP1) (make an attractive) alternative to isolated primary macrophages and can be used for assaying anti-leishmanial activity of different compounds against intracellular amastigotes.
Here, we present a parasite-rescue and transformation assay with differentiated THP1 cells infected in vitro with Leishmania donovani for screening pure compounds and natural products extracts and determining the efficacy against the intracellular Leishmania amastigotes. The assay involves the following steps: (1) differentiation of THP1 cells to non-dividing macrophages, (2) infection of macrophages with L. donovani metacyclic promastigotes, (3) treatment of infected cells with test drugs, (4) controlled lysis of infected macrophages, (5) release/rescue of amastigotes and (6) transformation of live amastigotes to promastigotes. The assay was optimized using detergent treatment for controlled lysis of Leishmania-infected THP1 cells to achieve almost complete rescue of viable intracellular amastigotes with minimal effect on their ability to transform to promastigotes. Different macrophage:promastigotes ratios were tested to achieve maximum infection. Quantification of the infection was performed through transformation of live, rescued Leishmania amastigotes to promastigotes and evaluation of their growth by an alamarBlue fluorometric assay in 96-well microplates. This assay is comparable to the currently-used microscopic, transgenic reporter gene and digital-image analysis assays. This assay is robust and measures only the live intracellular amastigotes compared to reporter gene and image analysis assays, which may not differentiate between live and dead amastigotes. Also, the assay has been validated with a current panel of anti-leishmanial drugs and has been successfully applied to large-scale screening of pure compounds and a library of natural products fractions (Tekwani et al. unpublished).

A quantitative analysis was done for both Digital-Image-Analysis-Direct-Counting-Assay and Parasite-Rescue-Transformation-Assay for 24, 48, 72 and 96 hr post-drug treatment
In the direct counting method, infection of L. donovani-infected macrophages (THP1 cells) was calculated by the following equation:
<img alt="Equation 1" fo:content-width="3.75in" fo:src="/files/ftp_upload/4054/4054eq1highres.jpg" src="/files/ftp_upload/4054/4054eq1.jpg" />
The amastigotes (determined by counting amastigotes nuclei)/100 transformed THP1 cells (determined by counting counted at THP1 cell nuclei) (Figure 7) is a more accurate measure to analyze the effect of different standard or test compounds than the percentage of infected THP1 cells, as reported in some previous papers, because this number is directly related to overall effect of compounds, either through a decrease in parasites in macrophage cells or total removal of parasite from the macrophage cells.Infection was calculated from digital images of infected THP1 cells treated with different standard drugs at different dilutions for various time intervals (Figure 10 and Table 1). The read-out for the Digital-Image-Analysis-Direct-Counting-Assay was amastigotes infection/100 transformed THP1 cells, while the read-out for the Parasite-Rescue-Transformation-Assay was relative fluorescence units (RFU), which is directly proportional to the number of live Leishmania amastigotes rescued from the infected macrophages and transform into promastigotes. The alamarBlue assay is routinely used for Leishmania promastigotes anti-leishmanial drug screening.
The assay was initially standardized and optimized for controlled lysis of Leishmania-infected THP1 cells. The objective was to optimize the conditions for detergent treatment, which yield almost complete lysis of THP1 cells with minimal effect on the viability of the rescued amastigotes. Figure 1 depicts a microscopic view of the complete assay protocol. Intact THP1 cells infected with Leishmania amastigotes can be seen in Figure 1A. Figure 1B shows lysis of the THP1 cells after detergent treatment. Figure 1C shows rescued Leishmania amastigotes, which have been partially transformed into promastigotes and Figure 1D shows almost complete transformation of amastigotes into promastigotes and their subsequent proliferation. The growth of these transformed promastigotes can be quantitatively monitored with addition of alamarBlue and measurement of fluorescence on a microplate reader. Treatment with NP-40 (Figure 2A) and Triton X-100 (Figure 2B) lysed the infected THP1 cells; however, it also influenced the viability and transformation of the rescued amastigotes. Treatment with Tween 20 (Figure 2C) and Tween 80 (Figure 2D) did not cause optimal lysis of THP1 cells resulting into an incomplete rescue of amastigotes as indicated by low numbers of transformed promastigotes. Treatment with 0.05% SDS for 30 sec (Figure 2E) yielded almost complete lysis of Leishmania-infected THP1 cells and did not affect viability and transformation of rescued amastigotes. Further optimization showed that treatment of cells with 0.05% SDS for 20-30 sec yielded highest rescue of viable Leishmania amastigotes (Figure 2F). In subsequent experiments, treatment with 0.05% SDS for 30 sec was used. Procedure for SDS treatment is the same for single or multiple plates. In multiple plates, SDS treatment was implemented column by column with a multichannel pipette. Serum-free medium was removed from all 8 wells of one column of the plate and 20 &mu;l of 0.05% SDS was added in 8 wells of the same column and diluted after 30 sec with RPMI-1640 with 10% FBS. During initial standardization of the assay, the plates were checked under the microscope for non-internalized promastigotes. A minimum of five washings were necessary for removal of parasites before step 5 of treatment of infected macrophage cells with standard compounds and three washings were necessary before step 7 of SDS treatment. Thus, the cells were washed 8 times and no visible non-internalized promastigotes remained before control lysis of infected THP1 cells.
Digital Image Analysis and Direct Counting
The digital images of Leishmania-infected THP1 cells were captured on Nikon Eclipse 90i fluorescent microscope after staining with SYBR Green I. Both macrophage nuclei and intracellular Leishmania nuclei with characteristic kinetoplast DNA were observed under the fluorescent filters (Figure 3). Further, the images of infected THP1 cells were also captured under DIC. When both the images were merged, the outlines of THP1 cells with intracellular amastigotes were seen more clearly (Figure 4). The ImageJ software was used to analyze these images. ImageJ is a public domain, Java-based, image-processing program developed at the National Institutes of Health (<a href="http://rsb.info.nih.gov/ij/download.html" target="_blank">http://rsb.info.nih.gov/ij/download.html</a>). ImageJ has been designed with an open architecture that provides extensibility via Java plugins and recordable macros. Custom acquisition, analysis and processing plugins can be developed using ImageJ's built-in editor and a Java compiler. For differential counting of THP1 cells nuclei and parasite nuclei by ImageJ, the image was opened in ImageJ. Cell counter was found in Analyze option in plugin of the Software. The image was initialized and cell counter type 1 was selected for THP1 cell nuclei and cell counter type 2 was selected for parasite nuclei (Figure 3). Differential counting was done for at least 200 THP1 cell nuclei and the intracellular amastigotes present in these THP1 cell nuclei. A comparison of parasite-rescue assay and image analysis method was made for evaluating the infectivity of THP1 cells with different macrophage:promastigotes ratios (Figure 4). Figure 5 represents the differential infectivity in THP1 cells at different macrophage:promastigote ratios. Both the methods showed comparable results and the macrophage:promastigote ratio of 1:10 yielded optimum and reproducible infectivity.
Once the conditions for parasite-rescue/transformation assay and the digital image analysis were optimized, the utility of these assays was evaluated for anti-leishmanial drug screening. The Leishmania-infected THP1 cells were treated with different concentrations of standard anti-leishmanial drugs namely Amphotericin B, Pentamidine and Miltefosine for different time intervals ranging from 24 to 96 hr. The experiment for parasite rescued/transformation assay was done in triplicate and the experiment for direct cells counting method was done in duplicate. Figure 6 shows microscopic images of the control uninfected, control infected untreated and Leishmania-infected, treated THP1 cells. The dose response curves were prepared from the parasite-rescue and transformation assay (concentration of the drug vs. transformed parasites) and the image analysis assay (number of amastigotes/100 THP1 cells) (Figures 7-9). The IC50 of the drugs were computed by ExcelFit and are presented in Table 1. Digital-Image-Analysis-Direct-Counting-Assay and Parasite-Rescue-Transformation-Assay showed comparable results. Digital-Image-Analysis-Direct-Counting-Assay was less optimal during the early time points of 24 and 48 hr drug treatments, while the Parasite-Rescue-Transformation-Assay showed results more consistent with the reported values at all the time points during the 24-96 hr after drug treatments. This difference in results with Digital-Image-Analysis-Direct-Counting-Assay and Parasite-Rescue-Transformation-Assay may be due to presence of non-viable amastigotes during early periods of drug treatment in Digital-Image-Analysis-Direct-Counting-Assay.
<img alt="Figure 1" src="/files/ftp_upload/4054/4054fig1.jpg" /> 
 Figure 1. A microscopic view of the Leishmania donovani amastigotes rescue and transformation to promastigotes. A - Adherent THP1 cells infected with Leishmania amastigotes; B - Adherent, infected THP1 cells after controlled lysis C - Transformed Leishmania donovani promastigotes from the amastigotes rescued from infected THP1 macrophage cells D - Growth and proliferation of transformed Leishmania donovani promastigotes.
<img alt="Figure 2" fo:content-width="5in" fo:src="/files/ftp_upload/4054/4054fig2highres.jpg" src="/files/ftp_upload/4054/4054fig2.jpg" /> 
 Figure 2. Optimization of controlled lysis of infected THP1 cells to achieve maximum rescue of live Leishmania donovani amastigotes and their transformation to promastigotes. Analysis of lysis of THP1 cells and rescue of amastigotes from the Leishmania-infected THP1 cells with different detergents. Two concentrations (0.05% and 0.1%) of detergent and two time periods (30 sec and 60 sec) for treatment were tested. RFU = relative fluorescence units. Each bar represents the mean of duplicate observations. [A] NP-40 treatment caused lysis of THP1 cells and also affected the viability of the rescued amastigote parasites. [B] Triton X-100 treatment caused lysis of THP1 cells and also affected the viability of the rescued amastigote parasites [C] Tween 80 caused partial lysis of THP1 cells to rescue the amastigotes.[D] Tween 80 caused partial lysis of THP1 cells to rescue the amastigotes. [E] SDS treatment caused almost complete lysis of THP1 cells and did not affect viability of the rescued amastigotes at 0.05%/30 sec. [F] Treatment with 0.05% SDS for 20-30 sec caused almost complete lysis of THP1 cells and rescued viable parasite amastigotes to transform into promastigotes. <a href="https://www.jove.com/files/ftp_upload/4054/4054fig2large.jpg" target="_blank"> Click here to view larger figure</a>.
<img alt="Figure 3" src="/files/ftp_upload/4054/4054fig3.jpg" /> 
 Figure 3. Fluorescent digital image of a differentiated THP1 cell infected in vitro with Leishmania donovani amastigotes. The characteristic kDNA can also be seen with each parasite nucleus. The macrophage nucleus (mN) (1) and the parasite nuclei (pN) (2) can be differentially marked and differentially counted by ImageJ analysis software for quantitative evaluation of the infection. The quantification was done as number of amastigotes/100 THP1 cells.
<img alt="Figure 4" src="/files/ftp_upload/4054/4054fig4.jpg" /> 
 Figure 4. Comparison between Digital-Image-Analysis-Direct-Counting-Assay (lower panel) and Parasite-Rescue-Transformation-Assay (upper panel). The macrophage:promastigote ratio of 1:10 yielded optimal infection. Both showed comparable results. The parasite-rescue assay showed some background values. Each bar shows mean of duplicate values.
<img alt="Figure 5" src="/files/ftp_upload/4054/4054fig5.jpg" /> 
 Figure 5. THP1 cells infected with Leishmania promastigotes by different THP1:promastigote ratio. The quantitative results as number of amastigotes/100 THP1 cells are presented in Figure 4.
<img alt="Figure 6" fo:content-width="5in" fo:src="/files/ftp_upload/4054/4054fig6highres.jpg" src="/files/ftp_upload/4054/4054fig6.jpg" /> 
 Figure 6. Digital images (Fluorescent + DIC) of THP1 cells infected with Leishmania donovani amastigotes after treatment with standard anti-leishmanial drugs for different time periods. Results were quantified as number of amastigotes/100 THP1 cells and used to compute the percent growth compared to untreated controls and determine the IC50 values.
<img alt="Figure 7" fo:content-width="4.75in" fo:src="/files/ftp_upload/4054/4054fig7highres.jpg" src="/files/ftp_upload/4054/4054fig7.jpg" /> 
 Figure 7. Comparison of Digital-Image-Analysis-Direct-Counting-Assay and Parasite-Rescue-Transformation-Assay for anti-leishmanial drug screening (Amophotericin B). The infected macrophages were treated with different concentrations of standard anti-leishmanial drug for different periods. IC50 (&mu;g/ml) values were computed from the dose response curve by Excelfit. <a href="https://www.jove.com/files/ftp_upload/4054/4054fig7large.jpg" target="_blank"> Click here to view larger figure</a>.
<img alt="Figure 8" fo:content-width="4.75in" fo:src="/files/ftp_upload/4054/4054fig8highres.jpg" src="/files/ftp_upload/4054/4054fig8.jpg" /> 
 Figure 8. Comparison of Digital-Image-Analysis-Direct-Counting-Assay and Parasite-Rescue-Transformation-Assay for anti-leishmanial drug screening (Pentamidine). The infected macrophages were treated with different concentrations of standard anti-leishmanial drug for different periods. IC50 (&mu;g/ml) values were computed from the dose response curves by Excelfit. <a href="https://www.jove.com/files/ftp_upload/4054/4054fig8large.jpg" target="_blank"> Click here to view larger figure</a>.
<img alt="Figure 9" fo:content-width="4.5in" fo:src="/files/ftp_upload/4054/4054fig9highres.jpg" src="/files/ftp_upload/4054/4054fig9.jpg" /> 
 Figure 9. Comparison of Digital-Image-Analysis-Direct-Counting-Assay and Parasite-Rescue-Transformation-Assay for anti-leishmanial drug screening (Mitefosine). The infected macrophages were treated with different concentrations of standard anti-leishmanial drug for different time periods. IC50 (&mu;g/ml) values were computed from the dose response curves by Excelfit. <a href="https://www.jove.com/files/ftp_upload/4054/4054fig9large.jpg" target="_blank"> Click here to view larger figure</a>.
<table border="1">
	<tbody>
		<tr>
			<td rowspan="2">Test Drug</td>
			<td colspan="2">24 hra</td>
			<td colspan="2">48 hra</td>
			<td colspan="2">72 hra</td>
			<td colspan="2">96 hra</td>
		</tr>
		<tr>
			<td>IACAb</td>
			<td>PRTAc</td>
			<td>IACAb</td>
			<td>PRTAc</td>
			<td>IACAb</td>
			<td>PRTAc</td>
			<td>IACAb</td>
			<td>PRTAc</td>
		</tr>
		<tr>
			<td>Amphotericin B</td>
			<td>0.24&plusmn; 0.03</td>
			<td>0.17&plusmn; 0.01*</td>
			<td>0.12&plusmn; 0.04</td>
			<td>0.20&plusmn; 0.07</td>
			<td>0.06&plusmn; 0.01</td>
			<td>0.06&plusmn; 0.01</td>
			<td>0.11&plusmn; 0.03</td>
			<td>0.10&plusmn; 0.03</td>
		</tr>
		<tr>
			<td>Pentamidine</td>
			<td>&gt;10</td>
			<td>2.55&plusmn; 1.16*</td>
			<td>2.88&plusmn; 0.58</td>
			<td>1.43&plusmn; 0.91</td>
			<td>1.24&plusmn; 0.35</td>
			<td>1.52&plusmn; 0.16</td>
			<td>0.71&plusmn; 0.63</td>
			<td>0.98&plusmn; 0.33</td>
		</tr>
		<tr>
			<td>Miltefosine</td>
			<td>0.38&plusmn; 0.02</td>
			<td>0.19&plusmn; 0.08*</td>
			<td>0.24&plusmn; 0.06</td>
			<td>0.30&plusmn; 0.08</td>
			<td>0.36&plusmn;0.02</td>
			<td>0.16&plusmn; 0.06</td>
			<td>0.21&plusmn; 0.15</td>
			<td>0.17&plusmn; 0.10</td>
		</tr>
	</tbody>
</table>
Table 1. Comparison of Digital-Image-Analysis-Direct-Counting-Assay and Parasite-Rescue-Transformation-Assay for anti-leishmanial drug screening. The infected macrophages were treated with different concentrations of standard anti-leishmanial drug for different periods. IC50 (&mu;g/ml) values were computed from the dose response curves by Excelfit (Figures 7-9). aHours post drug treatment; bIACA = Image Analysis and Direct Counting Assay; cPRTA = Parasite-Rescue and Transformation Assay. Values given are IC50 (concentration of the drug causing 50% inhibition in parasite growth) as &mu;g/ml and are the mean &plusmn; S.D. of at least three experiments. * Statistically different (&lt;0.05) compared to IC50 values with IACA.

Watch this Scientific Journal Video about A Parasite Rescue and Transformation Assay for Antileishmanial Screening Against Intracellular Leishmania donovani Amastigotes in THP1 Human Acute Monocytic L

A Parasite Rescue and Transformation Assay for Antileishmanial Screening Against Intracellular Leishmania donovani Amastigotes in THP1 Human Acute Monocytic Leukemia Cell Line

A parasite-rescue and transformation assay with THP1 cells infected in vitro with Leishmania donovani has been optimized for anti-leishmanial drug screening. The assay involves differentiation of THP1 cells, infection with promastigotes, treatment with test drugs, controlled lysis of the infected macrophages, rescue of amastigotes, transformation to promastigotes and monitoring promastigote growth and proliferation with a fluorometric assay.

A Parasite Rescue and Transformation Assay for Antileishmanial Screening Against Intracellular Leishmania donovani Amastigotes in THP1 Human Acute Monocytic Leukemia Cell Line

Leishmaniasis is  one of the world's most neglected diseases, largely affecting the  poorest of the poor, mainly in developing countries. Over 350 million ...

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Research

JoVE Journal

Immunology and Infection

Cutaneous Leishmaniasis in the Dorsal Skin of Hamsters: a Useful Model for the Screening of Antileishmanial Drugs

Traditionally, hamsters are experimentally inoculated in the snout or the footpad. However in these sites an ulcer not always occurs, measurement of lesion size is a hard procedure and animals show difficulty to eat, breathe and move because of the lesion. In order to optimize the hamster model for cutaneous leishmaniasis, young adult male and female golden hamsters (Mesocricetus auratus) were injected intradermally at the dorsal skin with 1 to 1.5 x l07 promastigotes of Leishmania species and progression of subsequent lesions were evaluated for up to 16 weeks post infection. The golden hamster was selected because it is considered the adequate bio-model to evaluate drugs against Leishmania as they are susceptible to infection by different species. Cutaneous infection of hamsters results in chronic but controlled lesions, and a clinical evolution with signs similar to those observed in humans. Therefore, the establishment of the extent of infection by measuring the size of the lesion according to the area of indurations and ulcers is feasible. This approach has proven its versatility and easy management during inoculation, follow up and characterization of typical lesions (ulcers), application of treatments through different ways and obtaining of clinical samples after different treatments. By using this method the quality of animal life regarding locomotion, search for food and water, play and social activities is also preserved.

Optimization of the experimental hamster model for cutaneous leishmaniasis by intradermal injection of Leishmania promastigotes at the dorsal skin. This approach is useful during inoculation, follow-up, characterization of lesions, application of treatments and obtaining of clinical samples. Locomotion, search for food and water, play and social activities are preserved.

Traditionally, hamsters are experimentally inoculated in the snout or the footpad. However in these sites an ulcer not always occurs, measurement of ...

A Microscopic Phenotypic Assay for the Quantification of Intracellular Mycobacteria Adapted for High-throughput/High-content Screening

Despite the availability of therapy and vaccine, tuberculosis (TB) remains one of the most deadly and widespread bacterial infections in the world. Since several decades, the sudden burst of multi- and extensively-drug resistant strains is a serious threat for the control of tuberculosis. Therefore, it is essential to identify new targets and pathways critical for the causative agent of the tuberculosis, Mycobacterium tuberculosis (Mtb) and to search for novel chemicals that could become TB drugs. One approach is to set&#160;up methods suitable for the genetic and chemical screens of large scale libraries enabling the search of a needle in a haystack. To this end, we developed a phenotypic assay relying on the detection of fluorescently labeled Mtb within fluorescently labeled host&#160;cells using automated confocal microscopy. This in vitro assay allows an image based quantification of the colonization process of Mtb&#160;into the host and was optimized for the 384-well microplate format, which is proper for screens of siRNA-, chemical compound- or Mtb mutant-libraries. The images are then processed for multiparametric analysis, which provides read&#160;out inferring on the pathogenesis of Mtb&#160;within host cells.

Here, we describe a phenotypic assay applicable to the High-throughput/High-content screens of small-interfering synthetic RNA (siRNA), chemical compound, and Mycobacterium tuberculosis mutant libraries. This method relies on the detection of fluorescently labeled Mycobacterium tuberculosis within fluorescently labeled host&#160;cell using automated confocal microscopy.

Despite the availability of therapy and vaccine, tuberculosis (TB) remains one of the most deadly and widespread bacterial infections in the world. Since ...

Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and reproducibility of this assay can be considered unstable, either because of user&#39;s errors or because of the sensitivity of NK cells to experimental manipulation. To eliminate these issues, a workflow that reduces them to a minimum was established and is presented here. To illustrate, we obtained blood samples, at various time points, from runners (n = 4) that were submitted to an intense bout of exercise. First, NK cells are simultaneously identified and isolated through CD56 tagging and magnetic-based sorting, directly from whole blood and from as little as one milliliter. The sorted NK cells are removed of any reagent or capping antibodies. They can be counted in order to establish an accurate NK cell count per milliliter of blood. Secondly, the sorted NK cells (effectors cells or E) can be mixed with 3,3&#39;-Diotadecyloxacarbocyanine Perchlorate (DiO) tagged K562 cells (target cells or T) at an assay-optimal 1:5 T:E ratio, and analyzed using an imaging flow-cytometer that allows for the visualization of each event and the elimination of any false positive or false negatives (such as doublets or effector cells). This workflow can be completed in about 4 h, and allows for very stable results even when working with human samples. When available, research teams can test several experimental interventions in human subjects, and compare measurements across several time points without compromising the data&#39;s integrity.

This work describes an advanced workflow for the accurate and fast determination of NK (Natural Killer) cell count and NK cell cytotoxicity in human blood samples.

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and ...

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread of multi drug-resistant TB (MDR-TB), there is a real urgency to develop new therapeutic strategies against M. tuberculosis infections. Traditionally, compounds are evaluated based on their antibacterial activity under in vitro growth conditions in broth; however, results are often misleading for intracellular pathogens like M. tuberculosis since in-broth phenotypic screening conditions are significantly different from the actual disease conditions within the human body. Screening for inhibitors that work inside macrophages has been traditionally difficult due to the complexity, variability in infection, and slow replication rate of M. tuberculosis. In this study, we report a new approach to rapidly assess the effectiveness of compounds on the viability of M. tuberculosis in a macrophage infection model. Using a combination of a cytotoxicity assay and an in-broth M. tuberculosis viability assay, we were able to create a screening system that generates a comprehensive analysis of compounds of interest. This system is capable of producing quantitative data at a low cost that is within reach of most labs and yet is highly scalable to fit large industrial settings.

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

We have developed a modular high-throughput screening system for discovering novel compounds against Mycobacterium tuberculosis, targeting intracellular and in-broth growing bacteria as well as cytotoxicity against the mammalian host cell.

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread ...

Isolation of the Side Population in Myc-induced T-cell Acute Lymphoblastic Leukemia in Zebrafish

Heterogeneous cell populations, from either healthy or malignant tissues, may contain a population of cells characterized by a differential ability to efflux the DNA-binding dye Hoechst 33342. This "side population" of cells can be identified using flow cytometric methods after the Hoechst 33342 dye is excited by an ultraviolet (UV) laser. The side population of many cell types contains stem- or progenitor-like cells. However, not all cell types have an identifiable side population. Danio rerio, zebrafish, have a robust in vivo model of T-cell acute lymphoblastic leukemia (T-ALL), but whether these zebrafish T-ALLs have a side population is unknown. The method described here outlines how to isolate the side population cells in zebrafish T-ALL. To begin, the T-ALL in zebrafish is generated via the microinjection of tol2 plasmids into one-cell stage embryos. Once the tumors have grown to a stage at which they expand into more than half of the animal's body, the T-ALL cells can be harvested. The cells are then stained with Hoechst 33342 and examined by flow cytometry for side population cells. This method has broad applications in zebrafish T-ALL research. While there are no known cell surface markers in zebrafish that confirm whether these side population cells are cancer stem cell-like, in vivo functional transplantation assays are possible. Furthermore, single-cell transcriptomics could be applied to identify the genetic features of these side population cells.

Here, we describe a technique to isolate the side population cells from a zebrafish model of myc-induced T-cell acute lymphoblastic leukemia (T-ALL). This side population assay is highly sensitive and is described for zebrafish T-ALL, but it may be applicable to other malignant and non-malignant zebrafish cell types.

Heterogeneous cell populations, from either healthy or malignant tissues, may contain a population of cells characterized by a differential ability to ...

Quantification of Intracellular Growth Inside Macrophages is a Fast and Reliable Method for Assessing the Virulence of Leishmania Parasites

The lifecycle of Leishmania, the causative agent of leishmaniasis, alternates between promastigote and amastigote stages inside the insect and vertebrate hosts, respectively. While pathogenic symptoms of leishmaniasis can vary widely, from benign cutaneous lesions to highly fatal visceral disease forms depending on the infective species, all Leishmania species reside inside host macrophages during the vertebrate stage of their lifecycle. Leishmania infectivity is therefore directly related to its ability to invade, survive and replicate within parasitophorous vacuoles (PVs) inside macrophages. Thus, assessing the parasite&#39;s ability to replicate intracellularly serves as a dependable method for determining virulence. Studying leishmaniasis development using animal models is time-consuming, tedious and often difficult, particularly with the pathogenically important visceral forms. We describe here a methodology to follow the intracellular development of Leishmania in bone marrow-derived macrophages (BMMs). Intracellular parasite numbers are determined at 24 h intervals for 72 - 96 h following infection. This method allows for a reliable determination of the effects of various genetic factors on Leishmania virulence. As an example, we show how a single allele deletion of the Leishmania Mitochondrial Iron Transporter gene (LMIT1) impairs the ability of the Leishmania amazonensis mutant strain LMIT1/&#916;Lmit1 to grow inside BMMs, reflecting a drastic reduction in virulence compared to wild-type. This assay also allows precise control of experimental conditions, which can be individually manipulated to analyze the influence of various factors (nutrients, reactive oxygen species, etc.) on the host-pathogen interaction. Therefore, the appropriate execution and quantification of BMM infection studies provide a non-invasive, rapid, economical, safe and reliable alternative to conventional animal model studies.

Quantification of Intracellular Growth Inside Macrophages is a Fast and Reliable Method for Assessing the Virulence of Leishmania Parasites

All pathogenic Leishmania species reside and replicate inside macrophages of their vertebrate hosts. Here, we present a protocol to infect murine bone marrow-derived macrophages in culture with Leishmania, followed by precise quantification of intracellular growth kinetics. This method is useful for studying individual factors influencing host-pathogen interaction and Leishmania virulence.

The lifecycle of Leishmania, the causative agent of leishmaniasis, alternates between promastigote and amastigote stages inside the insect and vertebrate ...

A Suction Blister Protocol to Study Human T-cell Recall Responses In Vivo

Cutaneous antigen-recall models allow for studies of human memory responses in vivo. When combined with skin suction blister (SB) induction, this model offers accessibility to rare populations of antigen-specific T-cells representative of the cellular memory response as well as the cytokine microenvironment in situ.
This report describes the practical procedure of a cutaneous recall, an SB induction, and a harvest of antigen-specific T-cells. To exemplify the method, the tuberculin skin test is used for antigenic recall in individuals who, prior to this study, underwent a Bacillus Calmette-Gu&#233;rin vaccination against an infection with Mycobacterium tuberculosis. Finally, examples of multiplex and flow cytometric analyses of SB specimens are provided, illustrating high fractions of antigen-specific polyfunctional CD4+ T-cells available by this sampling method compared with cells isolated from the blood.
The method described here is safe and minimally invasive, provides a unique opportunity to study both innate and adaptive immune responses in vivo, and may be beneficial to a broad community of researchers working with cell-mediated immunity and human memory responses, in the context of vaccine development.

A Suction Blister Protocol to Study Human T-cell Recall Responses In Vivo

Here, we provide a demonstration of the suction blister cutaneous recall model. The model allows a simple access to study human in vivo adaptive immune responses, for instance in the context of vaccine development.

Cutaneous antigen-recall models allow for studies of human memory responses in vivo. When combined with skin suction blister (SB) induction, this model ...

Opsonophagocytic Killing Assay to Assess Immunological Responses Against Bacterial Pathogens

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental procedure in which phagocytic cells are co-cultured with bacterial units. The immune cells will phagocytose and kill the bacterial cultures in a complement-dependent manner. The efficiency of the immune-mediated cell killing is dependent on a number of factors and can be used to determine how different bacterial cultures compare with regard to resistance to cell death. In this way, the efficacy of potential immune-based therapeutics can be assessed against specific bacterial strains and/or serotypes. In this protocol, we describe a simplified OPKA that utilizes basic culture conditions and cell counting to determine bacterial cell viability after co-culture with treatment conditions and HL-60 immune cells. This method has been successfully utilized with a number of different pneumococcal serotypes, capsular and acapsular strains, and other bacterial species. The advantages of this OPKA protocol are its simplicity, versatility (as this assay is not limited to antibody treatments as opsonins), and minimization of time and reagents to assess basic experimental groups.

This opsonophagocytic killing assay is used to compare the ability of phagocytic immune cells to respond to and kill bacteria based on different treatments and/or conditions. Classically, this assay serves as the gold&#160;standard for assessing effector functions of antibodies raised against a bacterium as opsonin.

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental ...

Using Human Induced Pluripotent Stem Cell-derived Intestinal Organoids to Study and Modify Epithelial Cell Protection Against Salmonella and Other Pathogens

The intestinal &#8216;organoid&#8217; (iHO) system, wherein 3-D structures representative of the epithelial lining of the human gut can be produced from human induced pluripotent stem cells (hiPSCs) and maintained in culture, provides an exciting opportunity to facilitate the modeling of the epithelial response to enteric infections. In vivo, intestinal epithelial cells (IECs) play a key role in regulating intestinal homeostasis and may directly inhibit pathogens, although the mechanisms by which this occurs are not fully elucidated. The cytokine interleukin-22 (IL-22) has been shown to play a role in the maintenance and defense of the gut epithelial barrier, including inducing a release of antimicrobial peptides and chemokines in response to infection.
We describe the differentiation of healthy control hiPSCs into iHOs via the addition of specific cytokine combinations to their culture medium before embedding them into a basement membrane matrix-based prointestinal culture system. Once embedded, the iHOs are grown in media supplemented with Noggin, R-spondin-1, epidermal growth factor (EGF), CHIR99021, prostaglandin E2, and Y-27632 dihydrochloride monohydrate. Weekly passages by manual disruption of the iHO ultrastructure lead to the formation of budded iHOs, with some exhibiting a crypt/villus structure. All iHOs demonstrate a differentiated epithelium consisting of goblet cells, enteroendocrine cells, Paneth cells, and polarized enterocytes, which can be confirmed via immunostaining for specific markers of each cell subset, transmission electron microscopy (TEM), and quantitative PCR (qPCR). To model infection, Salmonella enterica serovar Typhimurium SL1344 are microinjected into the lumen of the iHOs and incubated for 90 min at 37 &#176;C, and a modified gentamicin protection assay is performed to identify the levels of intracellular bacterial invasion. Some iHOs are also pretreated with recombinant human IL-22 (rhIL-22) prior to infection to establish whether this cytokine is protective against Salmonella infection.

Using Human Induced Pluripotent Stem Cell-derived Intestinal Organoids to Study and Modify Epithelial Cell Protection Against Salmonella and Other Pathogens

Human induced pluripotent stem cell (hiPSC)-derived intestinal organoids offer exciting opportunities to model enteric diseases in vitro. We demonstrate the differentiation of hiPSCs into intestinal organoids (iHOs), the stimulation of these iHOs with cytokines, and the microinjection of Salmonella Typhimurium into the iHO lumen, enabling the study of an epithelial invasion by this pathogen.

The intestinal &#8216;organoid&#8217; (iHO) system, wherein 3-D structures representative of the epithelial lining of the human gut can be produced from ...

Rescue of Recombinant Zika Virus from a Bacterial Artificial Chromosome cDNA Clone

The association of Zika virus (ZIKV) infection with neurological complications during the recent worldwide outbreak and the lack of approved vaccines and/or antivirals have underscored the urgent need to develop ZIKV reverse genetic systems to facilitate the study of ZIKV biology and the development of therapeutic and/or prophylactic approaches. However, like with other flaviviruses, the generation of ZIKV full-length infectious cDNA clones has been hampered due to the toxicity of viral sequences during its amplification in bacteria. To overcome this problem, we have developed a nontraditional approach based on the use of bacterial artificial chromosomes (BACs). Using this approach, the full-length cDNA copy of the ZIKV strain Rio Grande do Norte Natal (ZIKV-RGN) is generated from four synthetic DNA fragments and assembled into the single-copy pBeloBAC11 plasmid under the control of the human cytomegalovirus (CMV) immediate-early promoter. The assembled BAC cDNA clone is stable during propagation in bacteria, and infectious recombinant (r)ZIKV is recovered in Vero cells after transfection of the BAC cDNA clone. The protocol described here provides a powerful technique for the generation of infectious clones of flaviviruses, including ZIKV, and other positive-strand RNA viruses, particularly those with large genomes that have stability problems during bacterial&#160;propagation.

The recent epidemic of Zika virus highlights the importance of establishing reverse genetic approaches to develop vaccines and/or therapeutic strategies. Here, we describe the protocol to rescue an infectious recombinant Zika virus from a full-length cDNA clone assembled in a bacterial artificial chromosome under the control of the human cytomegalovirus immediate-early promoter.

The association of Zika virus (ZIKV) infection with neurological complications during the recent worldwide outbreak and the lack of approved vaccines ...