Sublingual Immunotherapy as an Alternative to Induce Protection Against Acute Respiratory Infections

The overall goal of this procedure is to use sublingual immunotherapy to boost the innate immune response in the lungs and comfort protection against lethal pneumococcal pneumonia. In mouse, the first step consists in administering a solution with the immunostimulatory agent flag into the sublingual mucosa of anesthetized mouse. Then a suspension containing a lethal dose of a genic strain of streptococcus pneumonia is instilled by intranasal root to anesthetized mice previously treated with flatline or saline as a control.

Broncho bilar lage is performed to analyze subpopulation infiltrating the airways and lung tissue is processed to obtain a single cell suspension for phenotypic characterization of the cell population. By multiparametric flow cytometry mandibular, an accessory mandibular lymph nodes as well as sublingual tissue can also be processed to perform flow cytometry or other downstream analysis. Cell populations infiltrating the airways.

Lungs, or sublingual mucosa can be identified by immune phenotyping and multiparametric flow cytometry Immune activation in different tissues after sublingual immunotherapy can be also analyzed by studying the transcription profile by realtime PCR. Welcome to the Laboratory for Vaccine Research at the Department of Biotechnology, we are allocating the school of medicine, university of U.One of the main interest of our lab is to generate new strategies to of immune intervention against respiratory infections. The internasal route has been successfully used for the delivery of immunostimulatory agents to treat or prevent lung infection or different a theology.

We have shown that intranasal delivery lin, the component of bacterial gel can be effectively used to broken ongoing lung infection caused by STO mice. Although the intranasal root normally requires low dose of the therapeutic agent and induces strong response in the respiratory tract, it has some drawbacks like the possibility of central nervous system toxicity and facia paralysis associated with absorption of immunostimulatory compounds across the oratory nerves. Hence, we have set up a model of UAL immunotherapy as an alternative to induce protective immunity against pneumococcal pneumonia.

Sublingual immunotherapy offers a safer alternative compared to intranasal installation. It is non-invasive, painless and simple. Although it may require higher doses of a therapeutic agent, it can be used to deliver NA or pharmaceutical products to ultimately modulate the immune response in the length.

We will now show you how to perform sublingual administration in mice using flattering as a therapeutic agent. Subin immunotherapy with flattering can eventually protect mice from a lethal intranasal challenge with a streptococcus pneumonia by triggering an innate immune response into the lung. We will also address characterization of length, innate response during infection and after sublingual immunotherapy using flowy geometry and real time PCR.

Finally, we will show you how to collect local graining lymph nodes and sublingual tissue for further Analysis. After preparing the solution of the stimulatory agent to be tested, anesthetize the animals by administering 200 microliters of the Ketamine OSI solution by Interperitoneal Injection Pet the solution containing the immuno stimulant in a maximum volume of 10 microliters. Then take the mouse with your left hand and hold it using your thumb and index finger with your free hand.

Gently place the closed forceps under the tongue and hold it in place. Using your middle and ring finger, take the pipet and administrate the solution onto the floor of the mouth and dorsal side of the tongue. Remove the forceps and allow the mouth to rest for three to five minutes before putting it back into the cage.

Key features of this technique are the total volume used to treat the animals and the anesthesia. The small size of the oral cavity mice limits the maximum volume that can be applied to the sublingual ratio to 10 microliters. Once decided the dose, you have to adjust the concentration of the sample to that maximum volume.

Bigger volumes could result in undesired swallowing or aspiration of the solution. Anesthesia is crucial to avoid swallowing and to allow penetration of the substances through the epithelium. We advise you not to use inhale to anesthesia since the mice will wake up too soon.

Note that some injectable anesthetics might induce salivation in mice. If that happens, use a small piece of fit or paper to eliminate the excessive salivation before applying the solution with the therapeutic agent, the dose of the therapeutic agent should be adjusted to maximize its effect and testing several doses is highly recommended. If you previously use your compound by intranasal root, a dose five to 10 times higher would be a good starting point to test.

Test your therapeutic agent by the sublingual root. Note that absorption through the epithelium will be limited by Size of the molecule thaw An aliquot of previously prepared working stock of streptococcus pneumonia of known bacterial CFU number centrifuge for five minutes at 2, 500 Gs and discard the supernat Suspend In one milliliter of ster saline and centrifuge. Again, discard the supernatant and then rest.

Suspend the pellet in the appropriate volume of sterile saline to obtain a suspension of four times 10 to the five CFU in 50 microliters, which corresponds to the minimum bacterial dose of the particulate serotype used that causes 100%mortality in bulb C.Mice take 50 microliters of the bacterial suspension and carefully instill the total volume into the nostrils of a previously anesthetized mouse. Hold the mouse upright for two minutes and let it rest in dorsal position for two more minutes. After the challenge, confirmed the CFU numbers in the bacterial suspension by performing serial tenfold dilution of the suspension and plate them onto blood.

Agar plate Incubate overnight at 37 Degrees with 5%CO2 muco. Colonies of ethno can be distinguished by the green alo characteristic of alpha hemolytic bacteria. Count colony forming units and register the challenge dose use.

After euthanizing the animal, open the thoracic cavity all the way up to the neck. Make an incision along the front legs to expose the trachea and sublingual area. Identify the mandibular lymph nodes.

These usually appear as a whiteish oval shaped structures sitting over the salivary glands. Using the fine tip curve forceps, gently pull up the sublingual tissue and expose the dorsal side of the mouth floor. Take the mandibular lymph nodes by pulling up gently with the core thin tip forceps to release them from the tissue.

Place them in a tube containing C-R-P-M-I or nucleic acid preservative solution according to the downstream procedure. That would be carried out later. Now identify the smaller lymph nodes located deeper at both sides of the throat and take them out.

These are the accessory mandibular lymph nodes. Open the thoracic cavity to expose the lung and remove the thymus. Cut the inferior binna CVA and remove the excess of blood with an absorbent tissue to perform the broncho lavage.

First, expose the trachea. Then make a small incision with a scalpel and introduce a thin tip. Transfer peppe previously filled with one milliliter of PBS without calcium magnesium plus one millimolar EDA Carefully Instill and aspirate the total volume at least three times.

Then aspirate and transfer the cell suspension to a sterile 1.5 milliliter tube. Perfuse the lungs by injecting five milliliters of PBS without calcium magnesium plus one millimolar EDTA into the right ventricle of the heart to eliminate all the red blood cells and immune cells present in the lungs blood vessels. If perfusion was performed correctly, lungs color will shift from pink to white.

Remove The heart, take the perfuse lungs and place them in complete medium or nucleic acid preservative solution depending on the downstream analysis to be performed. If you wish to analyze the cell population in the sublingual mucosa, isolate the head of the animal and remove the salivary glands and attach soft tissue. Make an incision on each side of the mouth until reaching the mandible joint and separate inferior jaw together with the tongue and floor of the mouth.

Using a pair of blunt rat tooth forceps, pull up the tongue and make an incision where the base of the tongue meets the floor of the mouth. This area constitutes the sublingual mucosa. Cut And remove the tongue using a biopsy punch, forceps and scissors.

Cut out the sublingual mucosal tissue and placed it into a clean tube. Transfer the lungs to a 24 well plate and means them with a clean pair of scissors. Add One milliliter of digestion media pipetting up and down several times and incubate at 37 degrees and 5%CO2 for 40 minutes after incubation.

Pipet up and down again to make sure that most of the cells had been released from the tissue. Transfer the cell suspension onto a 40 micro mesh cell strainer and washed with five milliliters of PBAs. Five millimolar EDTA centrifuge at 2, 500 GS five minutes.

Reus suspend cells in one milliliter fax EDTA made up with PBS without calcium magnesium one millimolar EDTA and 1%fetal boing serum. Take an QU and make a twofold dilution of the cells with trip and blue and count Reus suspend cells to a concentrations of two times 10 to the seven cells per milliliter and add 50 microliters per tube. Add 50 microliters of a two times antibody mix to each tube for immuno phenotypic analysis.

Incubate for 30 minutes on ice in the dark after washing three times with facts. EDTA resuspend the cells in 200 microliters of facts EDTA and analyze in a cytometer same procedure can be used to analyze cell population in sublingual tissue by using digestion media containing this space. Collagenase and DS one sublingual immunotherapy using purified flatline, an agonist of TLR five and LC four induced significant upregulation of the mRNA levels of chemokines and cytokines involved in neutrophil recruitment in the lungs.

This was also observed during infection. PMN recruitment into the lungs was confirmed by flow cytometry. Sublingual treatment with TLR five agonists correlated with increased survival of the animals and protection against pneumococcal pneumonia.

We have shown that sublingual immunotherapy is an effective means to induce protection in the lines against invasive PNE pneumococcal pneumonia. This was achieved by sublingual installation of lain, a tailor five agonists that is not produced by histo pneumonia. Sublingual installation of lain induced upregulation of the chemokine C Xcl L one in the lines, as well as infiltration of neutrophils.

A type of cells that are crucial to eliminating pneumococcus sublingual immunotherapy with TLR agonists could be used to treat infections alone or in combination with current therapies like antibiotic treatment in order to prevent bacterial dissemination and the development of severe forms of infection. Sublingual root can also be used to deliver vaccine and it has been shown to be effective for inducing mucosal as well as systemic protective immunity. This strategy is of particular interest in the case of respiratory infections and especially for bro pneumonias since it could not only prevent the basic disease, but also asal colonization blocking the bacteria at the sport of entry.

Overall, we believe that the sublingual route is an attractive alternative to develop new strategies to treat or prevent respiratory infections. We encourage you to try this technique.