This study was funded by Key Program of Chongqing Natural Science Foundation (No. cstc2020jcyj-zdxmX0027) and Chinese National Natural Science Foundation Project (No. 32270988).

The animal experiments were performed in accordance with the Guide to the Use and Care of Laboratory Animals and approved by the Laboratory Animal Welfare and Ethics Committee of the Third Military Medical University.
1. Preparation of nanoemulsions (NEs)
<ol>
	<li>For aqueous phase preparation, dissolve 0.15 g of polysorbate 80 in 28.2 mL of phosphate buffered saline (PBS) while stirring at 40 &#176;C.</li>
	<li>For oil phase preparation, use the oil phase formulation of the nanoemulsion shown in Table 1. Dissolve the sornitan trileate 85, DOTAP and RA in squalene while stirring at 40 &#176;C.</li>
	<li>For primary O/W emulsion preparation, stir the oil phase magnetically at 1400 rpm while adding the aqueous slowly and dropwise at a rate of 1 mL/min. Pre-emulsify the mixture using a high shear emulsifier at 30,000 rpm for 6 min.</li>
	<li>For high pressure homogenization (HPH), treat the primary O/W emulsion prepared above with a high-pressure homogenizer for 12 cycles at 900 bar to obtain a homogeneous nano-emulsion in the 160-190 nm range.</li>
	<li>At the end of the process, collect the emulsion in a 50 mL flask and filter-sterilize the emulsion through a 0.2 &#181;m PES filter membrane.</li>
	<li>Connect the flask to the Schlenk Line through the catheter, rotating the three-way stopcock to connect the system to the vacuum tube and turn on the vacuum pump to create vacuum in the flask. Then, rotating the three-way stopcock, connect the system to the Argon tube and fill it with Argon. Repeat this step 3x to ensure the flask is filled with Argon.</li>
	<li>Replace the cork and seal with a paraffine film, wrap the flask in tin foil and store it at 4 &#176;C.</li>
</ol>
2. Characterization of the nanoemulsions
<ol>
	<li>Particle size and zeta potential detection
	<ol>
		<li>Switch ON the instrument and wait 30 min for the laser light source to stabilize.</li>
		<li>Dilute the NE 50-fold with ultrapure water and add 1 mL of sample to the corresponding sample cell.</li>
		<li>To measure the particle size, set the temperature to 25 &#176;C, select water as the dispersing medium, and select disposable plastic dishes as the measuring cell. Load the samples and measure automatically. Report the mean value of three repeated measurements for each sample as the final result.</li>
		<li>For measurement of zeta potential, set the temperature to 25 &#176;C. Due to the choice of the aqueous phase as the dispersion medium, select the Smoluchowsi model, and the folded capillary cell as the measuring cell. Load the samples and measure automatically. Report the mean of three replicate measurements for each sample as the final result.</li>
	</ol>
	</li>
	<li>Determination of encapsulation rate and drug loading rate
	<ol>
		<li>To determine the amount of RA loaded in the NE, use absolute ethanol to demulsify and extract the RA from the NE. To 5 &#181;L of sample, add 995 &#181;L of ethanol and determine the RA content of the solution using a spectrophotometer at 355 nm. Compare the absorbance to a standard curve. Use the following equation: 
		Encapsulation = actual RA load/weight of drug added 
		Drug loading rate =actual RA loading/ sample&#39;s quality</li>
	</ol>
	</li>
</ol>
3. Immunization procedures and sample collection
<ol>
	<li>Immunization procedure and grouping
	<ol>
		<li>Group female Balb/C mice aged 6-8 weeks and weighing approximately 18-21 g in sets of 5 for immunization on day 0, day 14, day 28 according to the following experimental groups: (1) PBS group, (2) ovalbumin (OVA) group, (3) OVA+ NE-RA (OVA/ NE-RA) group, (4) OVA+CNE-RA(OVA/CNE-RA) group.</li>
		<li>Immunize all mice by intramuscular injection into the upper quadriceps muscles with 200 &#181;L of different vaccine formulations: 100 &#181;L of emulsion and 15 &#181;g OVA absorbed in 100 &#181;L of PBS, mixed before administration. Inject 100 &#181;L/hind leg. For mice in the control group administer PBS alone.</li>
		<li>Euthanasia: Euthanize all mice by an intraperitoneal injection of 100 mg/kg of 1% sodium pentobarbital 2 weeks after completion of the three immunizations.</li>
		<li>Sample collection and processing: After euthanasia, use scissors to cut open the chest of mice and insert a syringe directly into the heart to aspirate approximately 0.5 mL of whole blood. Allow the blood to stand for 2 h at room temperature and then centrifuge at 1800 x g for 5 min at 4 &#176;C. Collect serum, aliquot and store at -80 &#176;C for further analysis.</li>
		<li>Collect whole blood, spleen, vaginal lavage fluid (VLF), bronchoalveolar lavage fluid (BALF), nasal lavage fluid (NLF), and small intestinal lavage fluid (SILF).</li>
	</ol>
	</li>
	<li>Isolation of splenic lymphocytes
	<ol>
		<li>Soak mice in ethanol 75% (v/v) for a brief sterilization, transfer the mice to a clean bench. Cut the skin on the left abdomen with scissors, expose and remove the spleen.</li>
		<li>Place the spleen in a Petri dish containing 3 mL of PBS, grind and filter into a 15 mL centrifuge tube using a sieve (200 mesh, 70 &#181;m) and grinding rod.</li>
		<li>Centrifuge the cells at 650 x g for 5 min at room temperature. Discard the supernatant and suspend the precipitate with 3 mL of red blood cell lysis solution, incubate at room temperature for 5 min.</li>
		<li>Add 10 mL of PBS to the tube and shake well to terminate the reaction, then centrifuge it at 650 x g for 5 min at room temperature.</li>
		<li>Discard the supernatant and resuspend the cells in 10 mL of PBS, add 20 &#181;L of the cell dilution to the automated cell counter for cell counting.</li>
		<li>Centrifuge the cells at 650 x g for 5 min at room temperature. Discard the supernatant and dilute the cells to 1 x 106 cells/mL with 1640 complete medium (1% penicillin-streptomycin, 10% fetal bovine serum).</li>
	</ol>
	</li>
	<li>VLF collection: Rinse the vagina 4x with 75 &#181;L of 1%BSA/PBST, combine the lavage solution and collect in 1.5 mL centrifuge tubes, centrifuge at 5000 x g at 4 &#176;C for 20 min and collect the supernatant with a pipette and store at -80 &#176;C.</li>
	<li>BALF and NLF collection
	<ol>
		<li>Disinfect the mice after sacrifice with 75% (v/v) ethanol. Hold the mouse belly up and straighten the neck. Use scissors to make a longitudinal incision in the skin of the mouse neck and use forceps to separate the muscles and expose the trachea adequately.</li>
		<li>Cut the trachea through a small transverse opening and insert the hose toward the lungs, attach the syringe to the hose and inject 0.5 mL of PBS into the lungs and withdraw, repeat rinsing 3x and centrifuge at 650 x g at 4 &#176;C for 5 min, store the supernatant (BALF) at -80 &#176;C.</li>
		<li>Reverse the hose to the nasal cavity, attach the syringe to the hose and inject 0.5 mL of PBS, the liquid will flow through the nasal cavity into the 1.5 mL centrifuge tube. Aspirate the wash from the centrifuge tube and repeat the rinse 2x, then centrifuge at 650 x g at 4 &#176;C for 5 min, store the supernatant (NLF) at -80 &#176;C.</li>
	</ol>
	</li>
	<li>SILF collection
	<ol>
		<li>Prepare PBS solution containing 0.1 mg/mL trypsin inhibitor, 50 mM/L EDTA-2Na, 1 mM/L phenylmethylsulfonyl fluoride (PMSF) as the small intestine lavage solution. Stir at room temperature to dissolve and store at 4 &#176;C.</li>
		<li>Cut the small intestine open along the bowel tube and immerse in 3 mL of small intestinal lavage fluid. Mix by vertical shaking at 15 rpm for 30 min at 4 &#176;C. Centrifuge at 1440 x g at 4 &#176;C for 20 min and collect the supernatant with a pipette, then centrifuge at 5000 x g at 4 &#176;C for 20 min. Store the supernatant (SILF) at -80 &#176;C.</li>
	</ol>
	</li>
</ol>
4. Evaluation of OVA-specific antibody response after intramuscular administration
<ol>
	<li>Determine serum levels of IgG, subclasses of IgG1, IgG2a and the levels of specific sIgA in VLF, BALF, NLF and SILF by enzyme-linked immunosorbent assay (ELISA).</li>
	<li>For antigen coating, dilute OVA to 5 &#181;g/mL with coating buffer, and coat 96-well ELISA plates overnight at 4 &#176;C with 100 &#181;L of OVA solution per well.</li>
	<li>Wash ELISA plates 5x with PBST and block by incubation with 250 &#181;L of 1%BSA/PBST at 37 &#176;C for 2 h.</li>
	<li>Wash ELISA plates 5x with PBST, then add 100 &#181;L of the sample diluted as described below to be tested and incubate at 37 &#176;C for 1 h. Dilute serum, VLF, BALF, NLF with 0.5% BSA/PBST, dilute SILF with 20% fetal calf serum (FCS)/PBST.
	<ol>
		<li>Start by diluting the serum 1000x using a two-step dilution procedure: dilute 20 &#181;L of serum to 1 mL, then dilute 25 &#181;L of this diluted serum to 500 &#181;L. Use this dilution of serum for detection of IgG1, IgG2a.</li>
	</ol>
	</li>
	<li>Add 200 &#181;L of the serum diluted at 1000x to the first row of the coated plate and add 100 &#181;L of 0.5% BSA/PBST to all wells except the first row. Transfer 100 &#181;L from the first row to the second row and mix 10x with the pipette. Repeat this step up to row 8 and discard 100 &#181;L of liquid, the serum of different concentrations has been obtained for the detection of IgG.</li>
	<li>Perform a 1:1 dilution of BALF, NLF and SILF to detect IgA. Use the same dilution procedure for VLF as that for serum.</li>
	<li>Wash ELISA plates 5x with PBST. Add 100 &#181;L of HRP-conjugated goat anti-mouse IgG/IgG1/IgG2a/IgA (diluted 1:10000 with PBST) to the appropriate wells and incubate at 37 &#176;C for 40 min.</li>
	<li>Wash ELISA plates 5x with PBST, add 100 &#181;L of TMB ELISA substrate (highly sensitive), and transfer the plate to a place protected from light for 5 min. Immediately add 100 &#181;L of 450 nm stop solution for TMB substrate to stop the reaction.</li>
	<li>Measure the absorbance (OD) at 450 nm for each well and calculate the antibody titer by taking 2.1 times of the serum value of the blank group mouse as the positive response value.</li>
</ol>
5. ELISpot assay
<ol>
	<li>Follow the guidelines for ELISpot Plus, use Mouse IFN-&#947; (ALP) from the kit to perform the assays.</li>
	<li>Remove the plate from the sealed package and wash 4x with sterile PBS (200 &#181;L/well). Condition the plate with 1640 complete medium (200 &#181;L/well) and incubate for 30 min at room temperature.</li>
	<li>Remove the medium and add the adjusted concentration of lymphocyte suspension prepared in step3.2.2 (100 &#181;L/well) to the plate, then add 100 &#181;L of the stimulus i.e., 1640 complete medium containing 20 &#181;g/mL OVA257~264 or OVA323~339 or 1640 complete medium. Place the plate in a humidified incubator at 37 &#176;C with 5% CO2 for 48 h. Wrap the plate with aluminum foil to avoid evaporation.</li>
	<li>Discard the cell suspension and wash the plate 5x with PBS (200 &#181;L/well). Dilute the detection antibody (R4-6A2-biotin) to 1 &#181;g/mL in PBS containing 0.5% fetal calf serum (PBS-0.5% FCS). Add 100 &#181;L/well and incubate for 2 h at room temperature.</li>
	<li>Wash plate as in step 5.2. Dilute the streptavidin-ALP (1:1000) in PBS-0.5%FCS and add 100 &#181;L/well, incubate for 1 h at room temperature.</li>
	<li>Wash plate as in step 5.2. Filter the ready-to-use substrate solution (BCIP/NBT-plus) through a 0.45 &#181;m filter and add 100 &#181;L/well. Develop until distinct spots emerge.</li>
	<li>Stop color development by washing extensively in tap water, remove the soft plastic and leave the plate to dry.</li>
	<li>Inspect and count spots in an ELISpot reader.</li>
</ol>
6. Statistical analysis
<ol>
	<li>Analyze the differences among data of 4 groups by one-way ANOVA followed by Tukey multiple comparison post-test. Express all the results as mean &#177; SD. P value less than 0.05 was considered significant.</li>
</ol>

Evaluating Immunogenicity of Nanoemulsion&#45;Encapsulated Retinoic Acid in Mice

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The authors declare they have no conflicts of interest in this work.

In this protocol, we developed a cationic nanoemulsion-encapsulated retinoic acid to be used as an adjuvant to promote antigen-specific systemic and mucosal responses. Compared to traditional NE adjuvants, it has the following two advantages. First, in general, the surface of O/W NEs has a high negative charge, which makes it difficult to directly load antigens. Cationic NEs can effectively adsorb peptide or protein antigens and enhance the specific immunogenicity. Secondly, experience in traditional vaccine research has...

Adjuvants are often used to enhance the efficacy of a vaccine by stimulating the immune system to respond more strongly to the vaccine, thereby increasing immunity to a particular pathogen1. Nanoemulsion (NE) adjuvant refers to a colloidal dispersion system with thermodynamic stability by emulsifying a certain proportion of oil phase and aqueous phase to produce an emulsion in the form of water-in-oil (W/O) or oil-in-water (O/W)2. O/W nanoemulsion adjuvant can produce cytokines and chemokines at the injection site, induce the rapid aggregation and proliferation of important immune cells such as monocytes, neutrophils, and eosinophils, and enhance the immune response, and improve the immunogenicity of antigens3. Currently, three nanoemulsion adjuvants (MF59, AS03, and AF03) have been licensed for use in vaccines and have shown good safety and efficacy4.
However, mucosal immunity has been poorly addressed by the currently licensed adjuvant formulations in conventional parenteral vaccination5. Retinoic acid (RA) has been reported to induce intestinal homing of immune cells, but its low polarity, poor solubility in water, and poor light and thermal stability limit its use for robust enteric vaccination. Nanoemulsions offer opportunities to increase the bioavailability of highly lipophilic drugs, and the oil core of O/W emulsion adjuvants is suitable for dissolving non-polar substances such as RA6. Therefore, nanoemulsions can be used as carriers for RA in order to achieve the dual response effect of systemic immunity and mucosal immunity.
Compared to neutral or anionic delivery systems, cationic delivery systems have relatively efficient antigen encapsulation and delivery capabilities, which can enhance the immunogenicity of antigens7,8,9. The cationic surface charge of a variety of adjuvant systems is important for their adjuvant effects10,11,12. The cationic charge is an important factor in prolonging vaccine retention at the injection site, increasing antigen presentation and prolonging the stimulation of cellular immunity in the body12.
Based on the above considerations, we developed a cationic nanoemulsion to effectively co-deliver RA and antigens. The particle size and zeta potential of the nanoemulsion were determined using dynamic light scattering (DLS), and the systemic and mucosal immune responses of the nanoemulsion combined with OVA were evaluated by intramuscular injection13.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1640 medium</td>
			<td>GIBCO, USA</td>
			<td>C11875500BT</td>
			<td></td>
		</tr>
		<tr>
			<td>450 nm Stop Solution for TMB Substrate</td>
			<td>Abcam</td>
			<td>ab171529-1000 mL</td>
			<td></td>
		</tr>
		<tr>
			<td>Automated Cell Counter</td>
			<td>Countstar, China</td>
			<td>IC1000</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma-Aldrich, USA</td>
			<td>B2064-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge 5810 R</td>
			<td>Eppendorf, Germany</td>
			<td>5811000398</td>
			<td></td>
		</tr>
		<tr>
			<td>Danamic Light Scattering</td>
			<td>Malvern</td>
			<td>Zetasizer Nano S90</td>
			<td></td>
		</tr>
		<tr>
			<td>DOTAP</td>
			<td>CordenPharma, Switzerland</td>
			<td>O02002</td>
			<td></td>
		</tr>
		<tr>
			<td>ELISpot Plus: Mouse IFN-gamma (ALP)</td>
			<td>mabtech</td>
			<td>ab205719</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>GIBCO, USA</td>
			<td>10099141C</td>
			<td></td>
		</tr>
		<tr>
			<td>Full-function Microplate Reader</td>
			<td>Thermo Fisher Scientific, USA</td>
			<td>VL0000D2</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat Anti-Mouse IgG1(HRP)</td>
			<td>Abcam</td>
			<td>ab97240-1mg</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat Anti-Mouse IgA alpha chain (HRP)</td>
			<td>Abcam</td>
			<td>ab97235-1mg</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat Anti-Mouse IgG H&#38;L (HRP)</td>
			<td>Abcam</td>
			<td>Ab205720-500ug</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat&#160;Anti-Mouse IgG2a heavy chain (HRP)</td>
			<td>Abcam</td>
			<td>ab97245-1mg</td>
			<td></td>
		</tr>
		<tr>
			<td>High pressure homogenizer</td>
			<td>ATS</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MONTANE 85 PPI</td>
			<td>SEPPIC, France</td>
			<td>L12910</td>
			<td></td>
		</tr>
		<tr>
			<td>MONTANOX 80 PPI</td>
			<td>SEPPIC, France</td>
			<td>36372K</td>
			<td></td>
		</tr>
		<tr>
			<td>OVA257&#8211;264</td>
			<td>Shanghai Botai Biotechnology Co., Ltd.</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>OVA323-339</td>
			<td>Shanghai Botai Biotechnology Co., Ltd.</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffer saline</td>
			<td>ZSGB-bio</td>
			<td>ZLI-9061</td>
			<td></td>
		</tr>
		<tr>
			<td>Red Blood Cell Lysis Buffer</td>
			<td>Solarbio, China</td>
			<td>R1010</td>
			<td></td>
		</tr>
		<tr>
			<td>retinoic acid</td>
			<td>TCI, Japan</td>
			<td>TCI-R0064-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>Squalene</td>
			<td>Sigma, USA</td>
			<td>S3626</td>
			<td></td>
		</tr>
		<tr>
			<td>T10 basic Ultra-Turrax</td>
			<td>IKA, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TMB ELISA Substrate</td>
			<td>Abcam</td>
			<td>ab171523-1000ml</td>
			<td></td>
		</tr>
		<tr>
			<td>trypsin inhibitor</td>
			<td>Diamond</td>
			<td>A003570-0100</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Macklin, China</td>
			<td>9005-64-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultraviolet spectrophotometer</td>
			<td>Hitachi</td>
			<td>U-3900</td>
			<td></td>
		</tr>
	</tbody>
</table>

cationic nanoemulsion-encapsulated retinoic acid as an adjuvant to promote ova&#45;specific systemic and mucosal responses

Cationic nanostructures have emerged as an adjuvant and antigen delivery system that enhances dendritic cell maturation, ROS generation, and antigen uptake and then promotes antigen-specific immune responses. In recent years, retinoic acid (RA) has received increasing attention due to its effect in activating the mucosal immune response; however, in order to use RA as a mucosal adjuvant, it is necessary to solve the problem of its dissolution, loading, and delivery. Here, we describe a cationic nanoemulsion-encapsulated retinoic acid (CNE-RA) delivery system composed of the cationic lipid 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOTAP), retinoic acid, squalene as the oil phase, polysorbate 80 as surfactant, and sorbitan trioleate 85 as co-surfactant. Its physical and chemical properties were characterized using dynamic light scattering and a spectrophotometer. Immunization of mice with the mixture of antigen (ovalbumin, OVA) and CNE-RA significantly elevated the levels of anti-OVA secretory immunoglobulin A (sIgA) in vaginal lavage fluid and the small intestinal lavage fluid of mice compared with OVA alone. This protocol describes a detailed method for the preparation, characterization, and evaluation of the adjuvant effect of CNE-RA.

In total, four nanoemulsion formulations were prepared and characterized by their particle size (Figure 1), their zeta potential and their encapsulation efficiency as presented in Table 2. The particle size was concentrated around 160-190nm and the addition of DOTAP reversed the Zeta potential of nanoemulsion. OVA-specific serum IgG and its subgroup antibody level in serum were detected 2 weeks post third immunization. The nanoemulsion adjuvant vaccine significantly increase...

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Author Spotlight: Development and Evaluation of a Cationic Nanoemulsion&#45;Encapsulated Retinoic Acid System for Mucosal Vaccination

In this protocol, we developed a cationic nanoemulsion-encapsulated retinoic acid (RA) to be used as an adjuvant to promote antigen-specific systemic and mucosal responses. By adding the FDA-approved RA to the nanoemulsion, antigen-specific sIgA was promoted in the vagina and small intestine after intramuscular injection of the nanoemulsion.

Cationic Nanoemulsion-Encapsulated Retinoic Acid as an Adjuvant to Promote OVA&#45;Specific Systemic and Mucosal Responses

Cationic nanostructures have emerged as an adjuvant and antigen delivery system that enhances dendritic cell maturation, ROS generation, and antigen ...

We are working on a mucosal vaccine that produces an immune response as the set of infection, which has the potential to prevent infection and interrupt transmission. We have developed a catatonic nanoemulsion-encapsulated retinoic acid delivery system immunization with ovalbumin by intramuscular injection, which triggers both systemic and mucosal immune responses. In future studies, animal models of intestinal-related diseases can be used to further evaluate the effect and the mechanism of a cationic nanoemulsion-encapsulated retinoic acid in enhancing vaccine protection.

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JoVE Journal

Immunology and Infection

431 Views.  Third Military Medical University. We are working on a mucosal vaccine that produces an immune response as the set of infection, which has the potential to prevent infection and interrupt transmission. We have developed a catatonic nanoemulsion-encapsulated retinoic acid delivery system immunization with ovalbumin by intramuscular injection, which triggers both systemic and mucosal immune responses. In future studies, animal models of intestinal-related diseases can be used to further evaluate the effect and the mechanism of a...

Video: Author Spotlight: Development and Evaluation of a Cationic Nanoemulsion-Encapsulated Retinoic Acid System for Mucosal Vaccination

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

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

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

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

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

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

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. Although understanding the disease dynamics for each of these pathogens independently is vital, it is also important that the interactions between these pathogens are investigated and understood. 
We have developed a versatile in vitro model of HIV-malaria co-infection to study host immune responses to malaria in the context of HIV infection. Our model allows the study of secreted factors in cellular supernatants, cell surface and intracellular protein markers, as well as RNA expression levels. The experimental design and methods used limit variability and promote data reliability and reproducibility. 
All pathogens used in this model are natural human pathogens (Plasmodium falciparum and HIV-1), and all infected cells are naturally infected and used fresh. We use human erythrocytes parasitized with P. falciparum and maintained in continuous in vitro culture. We obtain freshly isolated peripheral blood mononuclear cells from chronically HIV-infected volunteers. Every condition used has an appropriate control (P. falciparum parasitized vs. normal erythrocytes), and every HIV-infected donor has an HIV uninfected control, from which cells are harvested on the same day. This model provides a realistic environment to study the interactions between malaria parasites and human immune cells in the context of HIV infection.

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Human co-infection is difficult to replicate in vitro. However, human malaria parasites can readily be cultured in vitro, as can freshly isolated human peripheral blood mononuclear cells naturally infected with HIV. This provides an excellent model for studying early immune responses to malaria parasites in the context of HIV co-infection.

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. ...

Experimental Infection with Listeria monocytogenes as a Model for Studying Host Interferon-&#947; Responses

L. monocytogenes is a gram-positive bacterium that is a cause of food borne disease in humans. Experimental infection of mice with this pathogen has been highly informative on the role of innate and adaptive immune cells and specific cytokines in host immunity against intracellular pathogens. Production of IFN-&#947; by innate cells during sublethal infection with L. monocytogenes is important for activating macrophages and early control of the pathogen1-3. In addition, IFN-&#947; production by adaptive memory lymphocytes is important for priming the activation of innate cells upon reinfection4. The L. monocytogenes infection model thus serves as a great tool for investigating whether new therapies that are designed to increase IFN-&#947; production have an impact on IFN-&#947; responses in vivo and have productive biological effects such as increasing bacterial clearance or improving mouse survival from infection. Described here is a basic protocol for how to conduct intraperitoneal infections of C57BL/6J mice with the EGD strain of L. monocytogenes and to measure IFN-&#947; production by NK cells, NKT cells, and adaptive lymphocytes by flow cytometry. In addition, procedures are described to: (1) grow and prepare the bacteria for inoculation, (2) measure bacterial load in the spleen and liver, and (3) measure animal survival to endpoints. Representative data are also provided to illustrate how this infection model can be used to test the effect of specific agents on IFN-&#947; responses to L. monocytogenes and survival of mice from this infection.

Experimental Infection with &lt;em&gt;Listeria monocytogenes&lt;/em&gt; as a Model for Studying Host Interferon-&amp;#947; Responses

This protocol describes how to inoculate C57BL/6J mice with the EGD strain of Listeria monocytogenes (L. monocytogenes) and to measure interferon-&#947; (IFN-&#947;) responses by natural killer (NK) cells, natural killer T (NKT) cells, and adaptive T lymphocytes post-infection. This protocol also describes how to conduct survival studies in mice after infection with a modified LD50 dose of the pathogen.

L. monocytogenes is a gram-positive bacterium that is a cause of food borne disease in humans. Experimental infection of mice with this pathogen has been ...

Analysis of Iophenoxic Acid Analogues in Small Indian Mongoose (Herpestes Auropunctatus) Sera for Use as an Oral Rabies Vaccination Biological Marker

The small Indian mongoose (Herpestes auropunctatus) is a reservoir of rabies virus (RABV) in Puerto Rico and comprises over 70% of animal rabies cases reported annually. The control of RABV circulation in wildlife reservoirs is typically accomplished by a strategy of oral rabies vaccination (ORV). Currently no wildlife ORV program exists in Puerto Rico. Research into oral rabies vaccines and various bait types for mongooses has been conducted with promising results. Monitoring the success of ORV relies on estimating bait uptake by target species, which typically involves evaluating a change in RABV neutralizing antibodies (RVNA) post vaccination. This strategy may be difficult to interpret in areas with an active wildlife ORV program or in areas where RABV is enzootic and background levels of RVNA are present in reservoir species. In such situations, a biomarker incorporated with the vaccine or the bait matrix may be useful. We offered 16 captive mongooses placebo ORV baits containing ethyl-iophenoxic acid (et-IPA) in concentrations of 0.4% and 1% inside the bait and 0.14% in the external bait matrix. We also offered 12 captive mongooses ORV baits containing methyl-iophenoxic acid (me-IPA) in concentrations of 0.035%, 0.07% and 0.14% in the external bait matrix. We collected a serum sample prior to bait offering and then weekly for up to eight weeks post offering. We extracted Iophenoxic acids from sera into acetonitrile and quantified using liquid chromatography/mass spectrometry. We analyzed sera for et-IPA or me-IPA by liquid chromatography-mass spectrometry. We found adequate marking ability for at least eight and four weeks for et- and me-IPA, respectively. Both IPA derivatives could be suitable for field evaluation of ORV bait uptake in mongooses. Due to the longevity of the marker in mongoose sera, care must be taken to not confound results by using the same IPA derivative during consecutive evaluations.

Analysis of Iophenoxic Acid Analogues in Small Indian Mongoose Herpestes Auropunctatus Sera for Use as an Oral Rabies Vaccination Biological Marker

We offered captive mongooses placebo oral rabies vaccine baits with ethyl or methyl iophenoxic acid as a biomarker and verified bait uptake using a novel liquid chromatography with tandem mass spectrometry (LC-MS/MS) method.

The small Indian mongoose (Herpestes auropunctatus) is a reservoir of rabies virus (RABV) in Puerto Rico and comprises over 70% of animal rabies cases ...

Isolation of Tonsillar Mononuclear Cells to Study Ex Vivo Innate Immune Responses in a Human Mucosal Lymphoid Tissue

Studying isolated cells from mucosa-associated lymphoid tissues (MALT) allows understanding of immune cells response in pathologies involving mucosal immunity, because they can model host-pathogen interactions in the tissue. While isolated cells derived from tissues were the first cell culture model, their use has been neglected because tissue can be hard to obtain. In the present protocol, we explain how to easily process and culture tonsillar mononuclear cells (TMCs) from healthy human tonsils to study innate immune responses upon activation, mimicking viral infection in mucosal tissues. Isolation of TMCs from the tonsils is quick, because the tonsils barely have any epithelium and yield up to billions of all major immune cell types. This method allows detection of cytokine production using several techniques, including immunoassays, qPCR, microscopy, flow cytometry, etc., similar to the use of peripheral mononuclear cells (PBMCs) from blood. Furthermore, TMCs show a higher sensitivity to drug testing than PBMCs, which needs to be considered for future toxicity assays. Thus, ex vivo TMCs cultures are an easy and accessible mucosal model.

In the present protocol, we explain how to easily process and culture tonsillar mononuclear cells from healthy humans undergoing partial surgical tonsillectomy to study innate immune responses upon activation, mimicking viral infection in mucosal tissues.

Studying isolated cells from mucosa-associated lymphoid tissues (MALT) allows understanding of immune cells response in pathologies involving mucosal ...

Assessing Respiratory Immune Responses to Haemophilus Influenzae

Haemophilus influenzae (Hi) is a prevalent bacterium found in a range of respiratory conditions. A variety of different assays/techniques may be used to assess the respiratory immune/inflammatory response to this bacterium. Flow cytometry and confocal microscopy are fluorescence-based technologies that allow detailed characterization of biological responses.&#160;Different forms of Hi antigen can be used, including cell wall components, killed/inactivated preparations, and live bacteria. Hi is a fastidious bacterium that requires enriched media but is generally easy to grow in standard laboratory settings. Tissue samples for stimulation with Hi may be obtained from peripheral blood, bronchoscopy, or resected lung (e.g., in patients undergoing surgery for the treatment of lung cancer). Macrophage and neutrophil function may be comprehensively assessed using flow cytometry with a variety of parameters measured, including phagocytosis, reactive oxygen species, and intracellular cytokine production. Lymphocyte function (e.g., T cell and NK cell function) may be specifically assessed using flow cytometry, principally for intracellular cytokine production. Hi infection is a potent inducer of extracellular trap production, both by neutrophils (NETs) and macrophages (METs). Confocal microscopy is arguably the most optimal way to assess NET and MET expression, which may also be used to assess protease activity. Lung immunity to Haemophilus influenzae can be assessed using flow cytometry and confocal microscopy.

Assessing Respiratory Immune Responses to Haemophilus Influenzae

Haemophilus influenzae induces inflammation in the respiratory tract. This article will focus on the use of flow cytometry and confocal microscopy to define immune responses by phagocytes and lymphocytes in response to this bacterium.

Haemophilus influenzae (Hi) is a prevalent bacterium found in a range of respiratory conditions. A variety of different assays/techniques may be used to ...

Assessing Serum Neutralization Against SARS-CoV-2 with Pseudotyped Viruses

Pseudotyped Viruses As a Molecular Tool to Monitor Humoral Immune Responses Against SARS-CoV-2 Via Neutralization Assay

Pseudotyped viruses (PVs) are molecular tools that can be used to study host-virus interactions and to test the neutralizing ability of serum samples, in addition to their better-known use in gene therapy for the delivery of a gene of interest. PVs are replication defective because the viral genome is divided into different plasmids that are not incorporated into the PVs. This safe and versatile system allows the use of PVs in biosafety level 2 laboratories. Here, we present a general methodology to produce lentiviral PVs based on three plasmids as mentioned here: (1) the backbone plasmid carrying the reporter gene needed to monitor the infection; (2) the packaging plasmid carrying the genes for all the structural proteins needed to generate the PVs; (3) the envelope surface glycoprotein expression plasmid that determines virus tropism and mediates viral entry into the host cell. In this work, SARS-CoV-2 Spike is the envelope glycoprotein used for the production of non-replicative SARS-CoV-2 pseudotyped lentiviruses.
Briefly, packaging cells (HEK293T) were co-transfected with the three different plasmids using standard methods. After 48 h, the supernatant containing the PVs was harvested, filtered, and stored at -80 &#176;C. The infectivity of SARS-CoV-2 PVs was tested by studying the expression of the reporter gene (luciferase) in a target cell line 48 h after infection. The higher the value for relative luminescence units (RLUs), the higher the infection/transduction rate. Furthermore, the infectious PVs were added to the serially diluted serum samples to study the neutralization process of pseudoviruses' entry into target cells, measured as the reduction in RLU intensity: lower values corresponding to high neutralizing activity.

Author Spotlight: Studying Host-Virus Interactions with Pseudotyped Viruses 

Pseudotyped viruses (PVs) are replication-defective virions that are used to study host-virus interactions under safer conditions than handling authentic viruses. Presented here is a detailed protocol that shows how SARS-CoV-2 PVs can be used to test the neutralizing ability of patients' serum after COVID-19 vaccination.

Pseudotyped viruses (PVs) are molecular tools that can be used to study host-virus interactions and to test the neutralizing ability of serum samples, in ...

Quantifying NET Formation Using Dual-Dye Live Cell Analysis

Real-Time High Throughput Technique to Quantify Neutrophil Extracellular Traps Formation in Human Neutrophils

Neutrophils are myeloid-lineage cells that form a crucial part of the innate immune system. The past decade has revealed additional key roles that neutrophils play in the pathogenesis of cancer, autoimmune diseases, and various acute and chronic inflammatory conditions by contributing to the initiation and perpetuation of immune dysregulation through multiple mechanisms, including the formation of neutrophil extracellular traps (NETs), which are structures crucial in antimicrobial defense. Limitations in techniques to quantify NET formation in an unbiased, reproducible, and efficient way have restricted our ability to further understand the role of neutrophils in health and diseases. We describe an automated, real-time, high-throughput method to quantify neutrophils undergoing NET formation using a live cell imaging platform coupled with a membrane permeability-dependent dual-dye approach using two different DNA dyes to image intracellular and extracellular DNA. This methodology is able to help assess neutrophil physiology and test molecules that can target NET formation.

Author Spotlight: Quantifying Neutrophil Extracellular Traps in Disease and Drug Screening Using Dual-Color Live-Cell Imaging

We present an automated high-throughput method to quantify neutrophil extracellular traps (NETs) utilizing the live cell analysis system, coupled with a membrane permeability-dependent dual-dye approach.

Neutrophils are myeloid-lineage cells that form a crucial part of the innate immune system. The past decade has revealed additional key roles that ...

Protocols for Analyzing Shigella Infection in Colonic Epithelial Cells

Epithelial Cell Infection Analyses with Shigella

The human-adapted enteric bacterial pathogen Shigella causes millions of infections each year, creates long-term growth effects among pediatric patients, and is a leading cause of diarrheal deaths worldwide. Infection induces watery or bloody diarrhea as a result of the pathogen transiting the gastrointestinal tract and infecting the epithelial cells lining the colon. With staggering increases in antibiotic resistance and the current lack of approved vaccines, standardized research protocols are critical to studying this formidable pathogen. Here, methodologies are presented to examine the molecular pathogenesis of Shigella using in vitro analyses of bacterial adherence, invasion, and intracellular replication in colonic epithelial cells. Prior to infection analyses, the virulence phenotype of Shigella colonies was verified by the uptake of the Congo red dye on agar plates. Supplemented laboratory media can also be considered during bacterial culturing to mimic in vivo conditions. Bacterial cells are then used in a standardized protocol to infect colonic epithelial cells in tissue culture plates at an established multiplicity of infection with adaptations to analyze each stage of infection. For adherence assays, Shigella cells are incubated with reduced media levels to promote bacterial contact with epithelial cells. For both invasion and intracellular replication assays, gentamicin is applied for various time intervals to eliminate extracellular bacteria and enable assessment of invasion and/or the quantification of intracellular replication rates. All infection protocols enumerate adherent, invaded, and/or intracellular bacteria by serially diluting infected epithelial cell lysates and plating bacterial colony forming units relative to infecting titers on Congo red agar plates. Together, these protocols enable independent characterization and comparisons for each stage of Shigella infection of epithelial cells to study this pathogen successfully.

Author Spotlight: Advancements in Understanding and Combatting Shigella Infections

The present protocol describes infection assays to interrogate Shigella adherence, invasion, and intracellular replication using in vitro epithelial cell lines.

The human-adapted enteric bacterial pathogen Shigella causes millions of infections each year, creates long-term growth effects among pediatric patients, ...