Support was provided by Becton Dickinson Technologies. Views expressed in this submission are those of the authors and do not purport to reflect official policy of the U.S. Government. Research was conducted in compliance with the Animal Welfare Act and other federal statutes and regulations relating to animals and experiments involving animals and adheres to principles stated in the Guide for the Care and Use of Laboratory Animals, National Research Council, 1996. The facility where this research was conducted is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International.

1. NALT Collection and Culturing
<ol>
	<li>Euthanize mice using approved IACUC guidance. Avoid use of inhalant anesthetics that may affect NALT. Transfer mice to an aseptic workspace or biosafety cabinet. Remove the lower jaw of the mouse and clean the upper palate area with alcohol and iodine wipes.</li>
	<li>Use a No. 11 surgical blade in surgical knife handle to carefully cut and excise the upper palate by following the inside contour of the mouse incisors and molar teeth.</li>
	<li>Gently peel back the palate with forceps, being careful not to tear the palate.</li>
	<li>This step is necessary to reduce contamination of cultures and is designed for processing multiple palates. Place palates into individual wells in the first column of a sterile 48-well plate pre-filled with 250 &mu;L of complete culture medium (37 &deg;C) consisting of RPMI 1640 supplemented with 10% fetal bovine serum, 100 &mu;g/mL streptomycin, 100 UI/mL penicillin, 50 &mu;g/mL gentamicin and 1 &mu;g/mL fungizone/amphotericin. Media should be carbonate buffered if palates are cultured in a 5% CO2/95% humid air incubator (37 &deg;C), or alternatively use 10 mM HEPES pH 7.4, for non-CO2 culture.</li>
	<li>To wash the palates, use forceps to move palates into each successive well in a row, carefully tapping the plate between washes, until the palate has undergone a total of eight washes.</li>
	<li>Transfer the palates into a new sterile 48-well plate containing 250 &mu;L of fresh, complete culture medium (37 &deg;C) in wells.</li>
	<li>Place the plates containing the palates into a 37 &deg;C incubator for culturing for the duration of the study.</li>
	<li>Collect samples for analysis by aseptically transferring 100 &mu;L of medium from culture wells into Eppendorf tubes every 24 hours, replacing with 100 &mu;L of fresh 37 &deg;C culture medium.</li>
	<li>Centrifuge the culture media samples at 380 x g for 10 minutes, 4 &deg;C, to pellet debris.</li>
	<li>Transfer the supernatant from the centrifuged sample to fresh Eppendorf tubes and store at -20 &deg;C until ready to analyze.</li>
	<li>Antigen-specific IgG, IgM, and IgA, or secreted cytokines are measured in tissue-culture supernatants by standard enzyme-linked immunosorbent assays (ELISA).</li>
</ol>
2. Surgical Ablation of NALT
<ol>
	<li>Female BALB/c mice, 7 to 9 weeks of age, are suitable subjects, but other strains are also acceptable. Provide gel-like wet food three days prior to surgery to acclimate mouse to food and water substitute that will be administered post-operation.</li>
	<li>Administer one drop of Metacam pain relief medication (1.5 mg/ml or 0.05 mg/drop) into the oral cavity prior to surgery.</li>
	<li>Anesthetize mice using approved IACUC guidance, for example, with a ketamine, acepromazine, and xylazine mixture (KAX) and apply Puralube Vet Ointment onto eyes to prevent drying. Avoid use of inhalant anesthetics that may affect NALT. Anesthesia can be confirmed by the absence of reflex response to a gentle pinching of the toes.</li>
	<li>Administer 1 mL saline (0.9% sodium chloride injection USP) subcutaneously between shoulder blades using a 22-28 gauge needle and syringe to prevent potential dehydration of the mouse after surgery. Also administer 0.1 mL of Respiram subcutaneously between shoulder blades using a 22-28 gauge needle and syringe to promote healthy respiration during and after surgery.</li>
	<li>Provide thermal support to the mouse during all subsequent manipulations.</li>
	<li>Place the anesthetized mouse in a supine position, and use two separate loops of surgical suture around the upper and lower incisors to gently pry open the mouth, exposing the upper palate.</li>
	<li>Use a No. 11 surgical blade to make an incision approximately 3 mm in length down the midline of the upper palate in the site of the NALT.</li>
	<li>Insert a 0.5 mm microcurette into the incision and gently scrape under the edges to disrupt the NALT.</li>
	<li>Cauterize the incision to stop bleeding with a straight fine loop on a thermal cautery unit.</li>
	<li>Continue thermal support while mouse recovers to full consciousness and activity.</li>
	<li>Provide mouse with saline subcutaneously up to three times a day, 1 mL per injection between shoulder blades, to prevent dehydration as necessary and oral pain medication once a day for 3 days post surgery. Determine dehydration by performing a skin tent test to check for skin turgor. Grasp the skin and fur between the shoulders blades so that it is tented up. If the skin quickly returns to the normal, previous position, the mouse is not dehydrated. If the skin remains elevated and moves slowly, the mouse is in a state of dehydration and requires saline. Provide wet food for at least three days after surgery to allow sufficient time for recovery.</li>
	<li>Prior to any experimental procedures, observe mice for 8-10 days by tracking weight gain. Poor weight recovery usually indicates incomplete palate healing, and these mice should be withdrawn from further studies.</li>
</ol>
3. Preparing NALT for Histology and Assessing the Success of NALT Surgery
<ol>
	<li>The success of NALT surgery must be ascertained by histology as described below. After completing all experimental studies, prepare mice for cranial collections by euthanizing using approved IACUC guidance. Avoid use of inhalant anesthetics that may affect NALT.</li>
	<li>Grasp the nape of the neck of the euthanized mouse. Remove the lower mandible from the rest of the skull by snipping through the condylar processes with scissors on both sides.</li>
	<li>Starting at the nape of the neck, remove the skin and fur from the skull by slowly peeling and cutting towards the ventral side of the cranium.</li>
	<li>Carefully remove skin around the nasal area and snip the skin off of the tip of the snout to remove completely.</li>
	<li>Detach the cranium from the vertebrae with a snip of the scissors.</li>
	<li>Insert scissors into the foramen magnum and cut halfway down the cranium along the midline to allow permeation of formalin into the soft tissues during fixation.</li>
	<li>Fix cranium in 10% neutral buffered formalin (NBF) for 24 hours at room temperature.</li>
	<li>To decalcify, place the sample and separate cassette, wrapped in gauze to prevent Rexyn from touching the bone, in bottle with the formic acid mixture. Incubate for 12 hours at room temperature, then rinse continuously under tap water for approximately 20 minutes.</li>
	<li>Trim the sample to the septum and past the eye orbits with a razor blade.</li>
	<li>Sample and cassette can be stored for short-term in 10% NBF.</li>
	<li>Place sample onto a tissue processor for overnight processing. This is done to remove water from the sample in preparation for paraffin embedding.</li>
	<li>Remove processed tissue, embed in a paraffin block with the snout down, and allow it to cool.</li>
	<li>Trim rough 15 &mu;m cross-sections of the block with an automated microtome, approaching the approximate location of the NALT, then continue cutting in 5 &mu;m sections for mounting onto glass slides in a 44.3 &deg;C water bath.</li>
	<li>Hematoxylin and eosin (H&amp;E) histology staining should be used to assess NALT ablation.</li>
</ol>
4. Collection of Biological Samples from Mice
4.1 Serum
<ol>
	<li>Carefully elevating body temperature with a heat lamp will increase blood flow. Collect blood by nicking the lateral tail vein with a surgical blade. Allow the blood to drip freely into a Microtainer serum separator tube.</li>
	<li>Apply gentle pressure to nick with absorbent material, such as gauze or towel, to stop blood flow.</li>
	<li>Allow the blood to clot in the Microtainers for 30 minutes, then centrifuge between 6-15 x 1,000 g for at least 90 seconds.</li>
	<li>Transfer separated serum from Microtainer into a clean Eppendorf tube for storage at -80 &deg;C.</li>
</ol>
4.2 Saliva
<ol>
	<li>Hold the anesthetized mouse vertically while pipetting 20-30 &mu;L sterile phosphate buffered saline (PBS) between the cheek and gum line. Collect the diluted saliva by directing pipette tip between cheek and gum line.</li>
	<li>Transfer the diluted saliva to a fresh tube containing 10 &mu;L of 2x protease inhibitor and store at -20 &deg;C.</li>
</ol>
4.3 Nasal secretions
<ol>
	<li>Euthanize mouse before collecting nasal secretions, using approved IACUC guidance. Avoid use of inhalant anesthetics that may affect NALT.</li>
	<li>Holding the mouse vertically, carefully pipette 30 &mu;L of sterile PBS in one nostril and collect rinse from other nostril.</li>
	<li>Transfer rinse to a fresh tube containing 10 &mu;L of 2x protease inhibitor and store at -20 &deg;C.</li>
</ol>
4.4 Vaginal secretions
<ol>
	<li>Insert the tip of a micropipette into the opening of the vulva of a euthanized mouse, rinse the vagina with 50 &mu;L of sterile PBS and collect all fluid by micropipette.</li>
	<li>Transfer rinse to a fresh tube containing 10 &mu;L of 2x protease inhibitor and store at -20 &deg;C.</li>
</ol>
5. Antigen-specific antibodies or cytokines in the collected samples can be measured by ELISA or another quantitative method.
6. Representative Results
Figure 1 provides a general schematic of steps involved with processing the NALT-containing tissue for ex vivo analysis. In Figure 2 (A, B), the size of the palate is shown, as well as the location of the incision during ablation surgery (A), as indicated by the dotted line. The location of NALT are indicated by arrows in the premolar area on an excised hematoxylin-stained palate in Figure 2 (C), showing the parallel tissues.
Figure 3 presents steps of the NALT disruption surgery, showing exposure of the upper palate for access to NALT (A, B), ablation (C), and final cauterization of the incision (D). A typical H &amp; E cross-section of the nasal sinus area surrounding the NALT before surgery is shown in Figure 3E, while an image of NALT disruption by the microcurette directly after surgery appears in Figure 3F. Allowing sufficient time for recuperation from surgery, the incisions should be closed and the nasal cavity devoid of NALT (Figure 3G).
Typical experimental results obtained by using these techniques are shown in Figure 4, comparing tissue culture supernatants and biological samples from a study of a staphylococcal subunit vaccine (STEBVax). Mice were administered STEBVax by intranasal (IN) or intraperitoneal (IP) routes. The vaccine was formulated with an adjuvant that activates the Toll-like receptor 4 pathway3,14, and controls were given only saline or vaccine without adjuvant. Cultured NALT obtained from experimental groups secreted antigen-specific immunoglobulins into medium that was measurable by ELISA. In this example (Figure 4A), the results indicate that the greatest amounts of IgA were released by NALT obtained from mice vaccinated IN with a subunit vaccine combined with adjuvant.
Biological samples (such as serum, saliva, nasal secretions, vaginal secretions) acquired from control or NALT-free mice can be used to profile the in vivo immune response to nasal antigens for comparison with the tissue culture results. In Figure 4B, IgA and IgG responses to IN vaccination were significantly decreased without functional NALT. Levels of antigen-specific IgA were generally greater than IgG in mucosal secretions (saliva, nasal washes) of vaccinated mice.
<img alt="Figure 1" src="/files/ftp_upload/3960/3960fig1.jpg" /> 
Figure 1. Schematic of NALT collection and ex vivo culturing.
<img alt="Figure 2" src="/files/ftp_upload/3960/3960fig2.jpg" /> 
Figure 2. Visualization of the mouse palate indicating the position of NALT and surgery incision. Size and location of upper palate with surgery incision denoted by dotted line (A); upper palate excised (B) or stained in hematoxylin (C) to view the parallel NALT in the premolar area of palate (stained dark purple on anterior side of palate). NALT indicated by arrows.
<img alt="Figure 3" src="/files/ftp_upload/3960/3960fig3.jpg" /> 
Figure 3. Surgical NALT disruption. Key stages of NALT disruption surgery: supine view of mouse upper palate before surgery (A); midline incision made on upper palate to access the NALT (B); microcurette probe inserted through midline incision to disrupt NALT structure integrity (C); cauterization of incision at conclusion of surgery (D). Microscopic images of nasal cavities, H&amp;E stained, before surgery (E); immediately after surgery (F); and a successfully healed, NALT-free mouse (G).
<img alt="Figure 4" src="/files/ftp_upload/3960/3960fig4.jpg" /> 
Figure 4. Vaccine-specific antibodies from cultured NALT, saliva, and nasal secretions collected from vaccinated mice. NALT-free or normal control mice were vaccinated IN or IP with STEBVax, and biological samples were collected. Antibody levels of triplicate samples were measured using ELISA. (A) NALT were removed from control mice (not surgically manipulated) and cultured to examine antibody responses. The vaccine-specific IgA response in culture for IN vaccinated mice was statistically different from controls (Student's t-test, p &le; 0.01, compared to no adjuvant or no vaccine). (B) NALT disruption substantially reduced specific antibody responses to IN vaccination. There were significant differences between specific antibody levels of NALT- and NALT+ groups (no surgery or control surgery) for all comparisons except saliva IgG results (Student's t-test, p&le; 0.05).

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No conflicts of interest declared.

We have presented collective methods for developing an animal model, obtaining biological samples, and assays for examining NALT-associated immune responses1-4. There are additional factors to consider during the performance of these methods. Standard sterile techniques for surgery and tissue culture should be followed. A combination of antibacterial and antifungal agents used during isolation and culture, as well as maintaining sterilized instruments, work area, and disinfected palates will reduce the risk of contamination. Saliva, nasal washes and similar mucosal secretion samples should be inspected for potential contamination with blood, as serum antibodies are generally found in higher concentrations. Further, mucosal secretions should only be slightly diluted for analysis because lower concentrations of antibodies are present in these samples compared to serum.
Mice must be kept warm directly after surgery to prevent potential anesthesia-induced hypothermia. Alternate resting mice on their sides during post-surgery recuperation to minimize irregular respiration. The surgical procedure is more efficient with three individuals working together to complete these tasks: one performing the surgery, one to assist in holding the mouth open, and one to provide post-surgical care as the mice recover from anesthesia. 
It is necessary to use H&amp;E staining of cranial cross-sections at the end of all experimental procedures or studies to verify the success of the surgery for each mouse. Possible surgery outcomes are: complete and bilateral NALT ablation, incomplete ablation, or intact NALT. Because not all surgeries will result in complete loss of the NALT, animals with residual or intact NALT can be used as internal controls. Another potential outcome is that the palate fails to completely heal, leaving an opening connecting the nasal and the oral cavities. Incomplete healing of the palate will result in low weight and failure to thrive, and these individuals should be removed from studies. 
Examining vaccine responses first with the mouse model will serve to establish a role for NALT in the intended study outcome (antibody response, survival, etc.). Surgically removing the NALT facilitates the determination of nasal contributions to local and systemic immunity. The surgical approach described here is the most direct method for obtaining a mouse model devoid of NALT. Select knock-out mouse models have been reported to lack NALT, but these animals also are deficient in cytokines or chemokines essential to the development of other secondary lymphoid tissues, and may harbor additional defects12,13. Further, the methods described here were developed for examining several aspects of immune responses originating within the nasal passages. Our experimental results are based on studies using the entire upper palate from the mouse for tissue culture, although it is possible that sections may be used. Finally, the cultured NALT model is useful for performing experiments completely in tissue culture.

<table border="1">
<tbody>
 <tr>
 <td>Name of the 			reagent</td>
 <td>Company</td>
 <td>Catalog 			number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Stainless 			Steel Sterile Surgical Blades, No. 11</td>
 <td>Miltex</td>
 <td>4-311</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Knife Handle, 			No. 3</td>
 <td>Miltex</td>
 <td>4-7</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>48-well Cell 			Culture Plates</td>
 <td>Costar</td>
 <td>3548</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>RPMI 1640</td>
 <td>Invitrogen</td>
 <td>11875-093</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Fetal Bovine 			Serum, Heat Inactivated</td>
 <td>Gibco/Invitrogen</td>
 <td>16000-044</td>
 <td>Final Conc: 			10% by volume in culture media </td>
 </tr>
 <tr>
 <td>Streptomycin 			Sulfate</td>
 <td>Sigma</td>
 <td>S9137</td>
 <td>Final Conc: 			100 &mu;g/mL</td>
 </tr>
 <tr>
 <td>Penicillin </td>
 <td>Sigma</td>
 <td>P7794</td>
 <td>Final Conc: 			100 UI/mL</td>
 </tr>
 <tr>
 <td>Gentamicin</td>
 <td>Sigma-Aldrich</td>
 <td>G1397-10ML</td>
 <td>Final Conc: 50 			&mu;g/mL</td>
 </tr>
 <tr>
 <td>Fungizone</td>
 <td>Gibco/Invitrogen</td>
 <td>15290-018</td>
 <td>Final Conc: 1 			&mu;g/mL</td>
 </tr>
 <tr>
 <td>HEPES</td>
 <td>Sigma-Aldrich</td>
 <td>H0887</td>
 <td>Final Conc: 10 			mM </td>
 </tr>
 <tr>
 <td>Eppendorf 			Microcentrifuge Tubes</td>
 <td>Eppendorf</td>
 <td>022364111</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Nutra-gel 			Cherry Flavored Mouse Wet Food</td>
 <td>Bio-Serve</td>
 <td>S4798-TRAY</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Metacam </td>
 <td>Boehringer 			Ingelheim</td>
 <td>601531000</td>
 <td>One drop of 			1.5 mg/mL Oral Suspension</td>
 </tr>
 <tr>
 <td>Ketamine</td>
 <td>Pfizer</td>
 <td>00856440301</td>
 <td>Final Conc: 			6.06 mg/mL</td>
 </tr>
 <tr>
 <td>Acepromazine</td>
 <td>Vedco</td>
 <td>VEDC207</td>
 <td>Final Conc: 			0.061 mg/mL</td>
 </tr>
 <tr>
 <td>Xylazine</td>
 <td>Lloyd</td>
 <td>4811</td>
 <td>Final Conc: 			0.667 mg/mL</td>
 </tr>
 <tr>
 <td>Puralube Vet 			Ointment</td>
 <td>Pharmaderm 			Animal Health</td>
 <td>1621</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>0.9% Sodium 			Chloride Injection USP</td>
 <td>Baxter</td>
 <td>2B1302</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>0.5 mm 			Microcurette</td>
 <td>Roboz</td>
 <td>RS-6350</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Thermal 			Cautery Unit</td>
 <td>Geiger</td>
 <td>150-S/150A-S</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>TCU 			Replacement Tip, Straight Fine Loop</td>
 <td>Geiger</td>
 <td>214</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Surgical 			Scissors </td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>10% Neutral 			Buffered Formalin</td>
 <td>Sigma</td>
 <td>HT501128</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Formic Acid</td>
 <td>Fisher</td>
 <td>A119P-4</td>
 <td>Mix 426 mL 			formic acid into 1047 mL tap water, then add 45 mL Rexyn 101 (H)</td>
 </tr>
 <tr>
 <td>Rexyn 101 (H)</td>
 <td>Fisher</td>
 <td>R231-500</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Tissue-Tek VIP 			E150/E300</td>
 <td>Sakura </td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Rotary 			Microtome</td>
 <td>Leica</td>
 <td>RM2255</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Mayer's 			Hematoxylin</td>
 <td>Sigma</td>
 <td>MHS16-500ML</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Gill No.1 			Hematoxylin</td>
 <td>Sigma</td>
 <td>GHS116</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Eosin B</td>
 <td>Sigma</td>
 <td>2853</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Microtainer 			Serum Separator</td>
 <td>BD Medical</td>
 <td>365956</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Phosphate 			Buffered Saline</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>100 mM 			NaH2PO4, 140 mM NaCl, pH 7.4</td>
 </tr>
 <tr>
 <td>Protease 			Inhibitor Cocktail, EDTA-free</td>
 <td>Thermo 			Scientific</td>
 <td>78415</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>

examining the role of nasopharyngeal-associated lymphoreticular tissue (nalt) in mouse responses to vaccines

The nasopharyngeal-associated lymphoreticular tissues (NALT) found in humans, rodents, and other mammals, contribute to immunity in the nasal sinuses1-3. The NALT are two parallel bell-shaped structures located in the nasal passages above the hard palate, and are usually considered to be secondary components of the mucosal-associated lymphoid system4-6. Located within the NALT are discrete compartments of B and T lymphocytes interspersed with antigen-presenting dendritic cells4,7,8. These cells are surrounded by an epithelial cell layer intercalated with M-cells that are responsible for antigen retrieval from the mucosal surfaces of the air passages9,10. Naive lymphocytes circulating through the NALT are poised to respond to first encounters with respiratory pathogens7. While NALT disappear in humans by the age of two years, the Waldeyer's Ring and similarly structured lymphatic organs continue to persist throughout life6. In contrast to humans, mice retain NALT throughout life, thus providing a convenient animal model for the study of immune responses originating within the nasal sinuses11.
Cultures of single-cell suspensions of NALT are not practical due to low yields of mononuclear cells. However, NALT biology can be examined by ex vivo culturing of the intact organ, and this method has the additional advantage of maintaining the natural tissue structure. For in vivo studies, genetic knockout models presenting defects limited to NALT are not currently available due to a poor understanding of the developmental pathway. For example, while lymphotoxin-&alpha; knockout mice have atrophied NALT, the Peyer's patches, peripheral lymph nodes, follicular dendritic cells and other lymphoid tissues are also altered in these genetically manipulated mice12,13. As an alternative to gene knockout mice, surgical ablation permanently eliminates NALT from the nasal passage without affecting other tissues. The resulting mouse model has been used to establish relationships between NALT and immune responses to vaccines1,3. Serial collection of serum, saliva, nasal washes and vaginal secretions is necessary for establishing the basis of host responses to vaccination, while immune responses originating directly from NALT can be confirmed by tissue culture. The following procedures outline the surgeries, tissue culture and sample collection necessary to examine local and systemic humoral immune responses to intranasal (IN) vaccination.

Watch this Scientific Journal Video about Examining the Role of Nasopharyngeal-associated Lymphoreticular Tissue NALT in Mouse Responses to Vaccines at JoVE.com

Examining the Role of Nasopharyngeal-associated Lymphoreticular Tissue NALT in Mouse Responses to Vaccines

Methods to examine contributions of the nasopharyngeal-associated lymphoreticular tissues (NALT) to nasal and systemic immune responses of mice to intranasal vaccines are described. We demonstrate a surgical procedure to establish a NALT-dependent mouse model and ex vivo cultures of extracted NALT.

Examining the Role of Nasopharyngeal-associated Lymphoreticular Tissue (NALT) in Mouse Responses to Vaccines

The nasopharyngeal-associated lymphoreticular tissues (NALT) found in humans, rodents, and other mammals, contribute to immunity in the nasal sinuses1-3. ...

examining-role-nasopharyngeal-associated-lymphoreticular-tissue-nalt

Universidad San Sebastian

Research

JoVE Journal

Immunology and Infection

25.7K Views.  U.S. Army Medical Research Institute of Infectious Diseases. Methods to examine contributions of the nasopharyngeal-associated lymphoreticular tissues (NALT) to nasal and systemic immune responses of mice to intranasal vaccines are described. We demonstrate a surgical procedure to establish a NALT-dependent mouse model and ex vivo cultures of extracted NALT.

Video: Examining the Role of Nasopharyngeal-associated Lymphoreticular Tissue NALT in Mouse Responses to Vaccines

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of Listeria, which results in systemic infection2. After injection, Listeria quickly disseminates to the spleen and liver due to uptake by CD8&alpha;+ dendritic cells and Kupffer cells3,4. Once phagocytosed, various bacterial proteins enable Listeria to escape the phagosome, survive within the cytosol, and infect neighboring cells5. During the first three days of infection, different innate immune cells (e.g. monocytes, neutrophils, NK cells, dendritic cells) mediate bactericidal mechanisms that minimize Listeria proliferation. CD8+ T cells are subsequently recruited and responsible for the eventual clearance of Listeria from the host, typically within 10 days of infection6.
Successful clearance of Listeria from infected mice depends on the appropriate onset of host immune responses6	. There is a broad range of sensitivities amongst inbred mouse strains7,8. Generally, mice with increased susceptibility to Listeria infection are less able to control bacterial proliferation, demonstrating increased bacterial load and/or delayed clearance compared to resistant mice. Genetic studies, including linkage analyses and knockout mouse strains, have identified various genes for which sequence variation affects host responses to Listeria infection6,8-14. Determination and comparison of infection kinetics between different mouse strains is therefore an important method for identifying host genetic factors that contribute to immune responses against Listeria. Comparison of host responses to different Listeria strains is also an effective way to identify bacterial virulence factors that may serve as potential targets for antibiotic therapy or vaccine design.
We describe here a straightforward method for measuring bacterial load (colony forming units [CFU] per tissue) and preparing single-cell suspensions of the liver and spleen for FACS analysis of immune responses in Listeria-infected mice. This method is particularly useful for initial characterization of Listeria infection in novel mouse strains, as well as comparison of immune responses between different mouse strains infected with Listeria. We use the Listeria monocytogenes EGD strain15 that, when cultured on blood agar, exhibits a characteristic halo zone around each colony due to &beta;-hemolysis1 (Figure 1). Bacterial load and immune responses can be determined at any time-point after infection by culturing tissue homogenate on blood agar plates and preparing tissue cell suspensions for FACS analysis using the protocols described below. We would note that individuals who are immunocompromised or pregnant should not handle Listeria, and the relevant institutional biosafety committee and animal facility management should be consulted before work commences.

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes is a model organism for studying immune responses and genetic susceptibility to intracellular bacteria in mice. This method enables one to measure bacterial load and generate single-cell suspensions of the liver and spleen from mice for FACS analysis to determine changes in immune cells due to Listeria infection.

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of ...

Intranasal Administration of Recombinant Influenza Vaccines in Chimeric Mouse Models to Study Mucosal Immunity

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the physiological mechanisms that mediate immune responses to vaccines perhaps due to the overall success of vaccination, which has reduced interest into the molecular and physiological mechanisms of vaccine immunity. However, several important human pathogens including influenza virus still pose a challenge for vaccination, and may benefit from immune-based strategies. 
Although influenza reverse genetics has been successfully applied to the generation of live-attenuated influenza vaccines (LAIVs), the addition of molecular tools in vaccine preparations such as tracer components to follow up the kinetics of vaccination in vivo, has not been addressed. In addition, the recent generation of mouse models that allow specific depletion of leukocytes during kinetic studies has opened a window of opportunity to understand the basic immune mechanisms underlying vaccine-elicited protection. Here, we describe how the combination of reverse genetics and chimeric mouse models may help to provide new insights into how vaccines work at physiological and molecular levels, using as example a recombinant, cold-adapted, live-attenuated influenza vaccine (LAIV). We utilized laboratory-generated LAIVs harboring cell tracers as well as competitive bone marrow chimeras (BMCs) to determine the early kinetics of vaccine immunity and the main physiological mechanisms responsible for the initiation of vaccine-specific adaptive immunity. In addition, we show how this technique may facilitate gene function studies in single animals during immune responses to vaccines. We propose that this technique can be applied to improve current prophylactic strategies against pathogens for which urgent medical countermeasures are needed, for example influenza, HIV, Plasmodium, and hemorrhagic fever viruses such as Ebola virus.

There is an overall lack of knowledge about how vaccines work. Here we propose the combined use of reverse genetics and bone marrow chimeric mice to gain insight into the early host immune responses to vaccines with a special focus on dendritic cells and T cell immunity.

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses

Airway inflammation is often associated with bacterial infections and represents a major determinant of lung disease. The in vivo determination of the pro-inflammatory capabilities of various factors is challenging and requires terminal procedures, such as bronchoalveolar lavage and the removal of lungs for in situ analysis, precluding longitudinal visualization in the same mouse. Here, lung inflammation is induced through the intratracheal instillation of Pseudomonas aeruginosa culture supernatant (SN) in transiently transgenized mice expressing the luciferase reporter gene under the control of a heterologous IL-8 bovine promoter. Luciferase expression in the lung is monitored by in vivo bioluminescent image (BLI) analysis over a 2.5- to 48-h timeframe following the instillation. The procedure can be repeated multiple times within 2 - 3 months, thus permitting the evaluation of the inflammatory response in the same mice without the need to terminate the animals. This approach permits the monitoring of pro- and anti-inflammatory factors acting in the lung in real time and appears suitable for functional and pharmacological studies.

An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses

The method described here allows for the visualization of IL-8 promoter-dependent inflammation activation in the lungs of mice through non-invasive bioluminescence imaging (BLI). The same animal can be subjected to BLI multiple times for up to two months from the time of delivery of the luciferase reporter construct.

Airway inflammation is often associated with bacterial infections and represents a major determinant of lung disease. The in vivo determination of the ...

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple condensation reactions. DHDDS (dehydrodolichyl diphosphate synthase) is a eukaryotic long-chain cis-PT (forming cis double bonds from the condensation reaction) that catalyzes chain elongation of farnesyl diphosphate (FPP, an allylic diphosphate) via multiple condensations with isopentenyl diphosphate (IPP). DHDDS is of biomedical importance, as a non-conservative mutation (K42E) in the enzyme results in retinitis pigmentosa, ultimately leading to blindness. Therefore, the present protocol was developed in order to acquire large quantities of purified DHDDS, suitable for mechanistic studies. Here, the usage of protein fusion, optimized culture conditions and codon-optimization were used to allow the overexpression and purification of functionally active human DHDDS in E. coli. The described protocol is simple, cost-effective and time sparing. The homology of cis-PT among different species suggests that this protocol may be applied for other eukaryotic cis-PT as well, such as those involved in natural rubber synthesis.

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

A simple protocol for overexpression and purification of codon-optimized, human cis-prenyltransferase, under non-denaturing conditions, from Escherichia coli, is described, along with an enzymatic activity assay. This protocol can be generalized for production of other cis- prenyltransferase proteins in quantity and quality suitable for mechanistic studies.

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. typhimurium bacteria are quickly detected and phagocytized by macrophages, and contained in vesicles known as phagosomes in order to be degraded. Isolation of S. typhimurium-containing phagosomes have been widely used to study how S. typhimurium infection alters the process of phagosome maturation to prevent bacterial degradation. Classically, the isolation of bacteria-containing phagosomes has been performed by sucrose gradient centrifugation. However, this process is time-consuming, and requires specialized equipment and a certain degree of dexterity. Described here is a simple and quick method for the isolation of S. typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin-streptavidin-conjugated magnetic beads. Phagosomes obtained by this method can be suspended in any buffer of choice, allowing the utilization of isolated phagosomes for a broad range of assays, such as protein, metabolite, and lipid analysis. In summary, this method for the isolation of S. typhimurium-containing phagosomes is specific, efficient, rapid, requires minimum equipment, and is more versatile than the classical method of isolation by sucrose gradient-ultracentrifugation.

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

We describe here a simple and quick method for the isolation of Salmonella typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin and streptavidin.

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

Immunization of Adult Zebrafish for the Preclinical Screening of DNA-based Vaccines

The interest in DNA-based vaccination has increased during the past two decades. DNA vaccination is based on the cloning of a sequence of a selected antigen or a combination of antigens into a plasmid, which enables a tailor-made and safe design. The administration of DNA vaccines into host cells leads to the expression of antigens that stimulate both humoral and cell-mediated immune responses. This report describes a protocol for the cloning of antigen sequences into the pCMV-EGFP plasmid, the immunization of adult zebrafish with the vaccine candidates by intramuscular microinjection, and the subsequent electroporation to improve intake. The vaccine antigens are expressed as green fluorescent protein (GFP)-fusion proteins, which allows the confirmation of the antigen expression under UV light from live fish and the quantification of expression levels of the fusion protein with ELISA, as well as their detection with a western blot analysis. The protective effect of the vaccine candidates is tested by infecting the fish with Mycobacterium marinum five weeks postvaccination, followed by the quantification of the bacteria with qPCR four weeks later. Compared to mammalian preclinical screening models, this method provides a cost-effective method for the preliminary screening of novel DNA-based vaccine candidates against a mycobacterial infection. The method can be further applied to screening DNA-based vaccines against various bacterial and viral diseases.

Here we describe a protocol for the immunization of the adult zebrafish (Danio rerio) with a DNA-based vaccine and demonstrate the validation of a successful vaccination event. This method is suitable for the preclinical screening of vaccine candidates in various infection models.

The interest in DNA-based vaccination has increased during the past two decades. DNA vaccination is based on the cloning of a sequence of a selected ...

Evaluation of Host-Pathogen Responses and Vaccine Efficacy in Mice

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a striking increase in life expectancy around the globe. Nonetheless, determining vaccine efficacy remains a challenge. Emerging evidence suggests that the current acellular vaccine (aPV) for Bordetella pertussis (B. pertussis) induces suboptimal immunity. Therefore, a major challenge is designing a next-generation vaccine that induces protective immunity without the adverse side effects of a whole-cell vaccine (wPV). Here we describe a protocol that we used to test the efficacy of a promising, novel adjuvant that skews immune responses to a protective Th1/Th17 phenotype and promotes a better clearance of a B. pertussis challenge from the murine respiratory tract. This article describes the protocol for mouse immunization, bacterial inoculation, tissue harvesting, and analysis of immune responses. Using this method, within our model, we have successfully elucidated crucial mechanisms elicited by a promising, next-generation acellular pertussis vaccine. This method can be applied to any infectious disease model in order to determine vaccine efficacy.

Here we present an elegant protocol for in vivo evaluation of vaccine effectiveness and host immune responses. This protocol can be adapted for vaccine models that study viral, bacterial, or parasitic pathogens.

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a ...

Bone Marrow Transplantation Platform to Investigate the Role of Dendritic Cells in Graft-versus-Host Disease

Allogeneic bone marrow transplantation (BMT) is an effective therapy for hematological malignancies due to the graft-versus-leukemia (GVL) effect to eradicate tumors. However, its application is limited by the development of graft-versus-host disease (GVHD), a major complication of BMT. GVHD is evoked when T-cells in the donor grafts recognizealloantigen expressed by recipient cells and mount unwanted immunological attacks against recipient healthy tissues. Thus, traditional therapies are designed to suppress donor T-cell alloreactivity. However, these approaches substantially impair the GVL effect so that the recipient&#39;s survival is not improved. Understanding the effects of therapeutic approaches on BMT, GVL, and GVHD, is thus essential. Due to the antigen-presenting and cytokine-secreting capacities to stimulate donor T-cells, recipient dendritic cells (DCs) play a significant role in the induction of GVHD. Therefore, targeting recipient DCs becomes a potential approach for controlling GVHD. This work provides a description of a novel BMT platform to investigate how host DCs regulate GVH and GVL responses after transplantation. Also presented is an effective BMT model to study the biology of GVHD and GVL after transplantation.

Graft-versus-host disease is a major complication after allogeneic bone marrow transplantation. Dendritic cells play a critical role in the pathogenesis of graft-versus-host disease. The current article describes a novel bone marrow transplantation platform to investigate the role of dendritic cells in the development of graft-versus-host disease and the graft-versus-leukemia effect.

Allogeneic bone marrow transplantation (BMT) is an effective therapy for hematological malignancies due to the graft-versus-leukemia (GVL) effect to ...

Highly Efficient Transfection of Primary Macrophages with In Vitro Transcribed mRNA

Macrophages are phagocytic cells specialized in detecting molecules of non-self origin. To this end, they are equipped with a large array of pattern recognition receptors (PRRs). Unfortunately, this also makes macrophages particularly challenging to transfect as the transfection reagent and the transfected nucleic acids are often recognized by the PRRs as non-self. Therefore, transfection often results in macrophage activation and degradation of the transfected nucleic acids or even in suicide of the macrophages. Here, we describe a protocol that allows highly efficient transfection of murine primary macrophages such as peritoneal macrophages (PM) and bone marrow-derived macrophages (BMDM) with mRNA in vitro transcribed from DNA templates such as plasmids. With this simple protocol, transfection rates of about 50-65% for PM and about 85% for BMDM are achieved without cytotoxicity or immunogenicity observed. We describe in detail the generation of mRNA for transfection from DNA constructs such as plasmids and the transfection procedure.

Macrophages, especially primary macrophages, are challenging to transfect as they specialize in detecting molecules of non-self origin. We describe a protocol that allows highly efficient transfection of primary macrophages with mRNA generated from DNA templates such as plasmids.

Macrophages are phagocytic cells specialized in detecting molecules of non-self origin. To this end, they are equipped with a large array of pattern ...