Cultivation of Heligmosomoides Polygyrus: An Immunomodulatory Nematode Parasite and its Secreted Products

Heligmosomoides polygyrus is a murine nematode with powerful immunomodulatory capabilities that closely resemble those of highly-prevalent human helminth infection. Here we describe a protocol for the long-term maintenance of the H. polygyrus lifecycle.

The overall goal of this procedure is to collect and analyze the molecules secreted by long-lived helmuth parasites that downregulate the immune responses of the host, thereby permitting parasites survival. This is accomplished by first growing the parasites in their natural habitat, the intestinal tract of mice. The second step is to isolate the mature parasites from the mice following euthanasia.

Next, the helmuth parasites are cultured in tissue culture, medium erupt to three weeks during which time they release copious quantities of excretory, secretory, or ES molecules. The final step is to recover and concentrate the released molecules from the culture supernatants into batches of ES for biological and biochemical testing. Ultimately, the concentrated supernatants can be tested directly on specific cell types in vitro to study changes in immune cell function or administered to mice to test their ability to suppress allergy and other disorders.

We first had the idea for this method when we noted that immune suppression was linked to live parasite infection indicating active and ongoing release of immunosuppressive molecules from live parasites. Though this method can provide insight into parasite infections, the molecules, we discover that suppress immunity are likely to be very effective in treating key diseases of the western world, such as allergy autoimmunity and inflammatory bowel disease. Visual demonstration of this method is critical as the steps to recover and process helmuth parasites are quite specialized and need particular attention to aseptic culture Technique.

Eight week old F1 mice will be infected with H polyus larvae by oral gavage for the lifecycle production of H polyus. Prepare H polyus L three larvae at 2000 per milliliter of distilled water as described in the accompanying protocol. Text to give 400 L three per mouse in 200 microliters prior to each infection.

A educate the larvae suspension by inverting it five times using a one milliliter syringe with a dedicated gavage needle with a rounded end aspirate 200 microliters of the larvae suspension. To perform oral gavage, restrain the mouse in an upright position by the scruff of the neck and gently pass the blunt gavage needle through the mouth and esophagus into the stomach. Adult worms and parasite eggs will be recovered from the mouse.

14 days after infection, 14 days after infection. Adult h polyus worms are collected from the mice using a modified bayamon apparatus prepared as shown here. To begin this procedure, wash the abdomen of the euthanized animal with 70%ethanol.

Cut the skin over the abdomen and to pull back to reveal the anterior abdominal wall. Make a midline incision to enter the peritoneal cavity. Remove the entire small and large intestine from the proximal duodenum to the distal rectum.

Place it in a dry petri dish. Straighten the gut along its entire length that excise the feces containing colon, and place this in a separate dish to recover eggs later. Next, identify the proximal 20 centimeters of small intestine that contains the adult worms.

It is characterized by the relatively thick wall of the duodenum and often has a red appearance due to the intraluminal worms. Excise this portion of the small intestine and place it into a petro dish with five milliliters of Hank's solution. Warm to 37 degrees Celsius using round ended scissors.

Open the worm filled proximal gut portion longitudinally and scrape down the inside of the gut lining with two glass slides. To remove the worms, discard the clean gut wall. Build the funnel with Hank's solution.

Staple a small muslin bag closed, and tip the worms from two Petri dishes into it. Arrange two muslin bags to each glass funnel and secure the bags with paperclips around the edge of the funnel. Place the apparatus in a 37 degrees Celsius incubator for one to two hours.

Halfway through the incubation, gently agitate the apparatus to dislodge debris from the gut preparation that may occlude the muslin filter. Take care to avoid spillage of debris outside the muslin bag as this will cause contamination at the final HES preparation. At the end of the incubation, carefully detach the collection test tube from the connecting rubber hose over a sink using a plastic pipette, transfer the worms into a 50 milliliter tube and allow the worms to settle with gravity.

Remove the media with a stripette. Add 40 milliliters of Hank's solution and allow the worms to settle Repeat five times for a total of six washes. Avoiding contamination of the haze culture is critical for the success of this protocol.

The worm culture must be kept sterile from this point onward. Move to a laina flow hood and wash the worm culture another six times in sterile Hank's solution, supplemented with antibiotics using a pipette and collect two samples of 20 microliters for counting the adult worms recovered. Approximately 50%of the quantity of inoculated larvae is expected to collect eggs at day 14 of infection to scrape feces out of the previously excised colon with forceps.

Next, mix the feces with granulated charcoal at a ratio of at least one to one. To achieve a consistency, just dam enough to a adhere to fill a paper. Smear a thin layer on the center of a piece of dampen filter paper in a Petri dish and place this in a humid light proof box to keep it dark for 12 to 14 days.

From day seven onwards, start collecting L three larvae a larvae. Form a ring around the edge of the filter paper. Lift the filter paper outta the Petri dish and use a pipette, a five milliliters of sterile water per plate to rinse the larvae that are left on the plate.

Transfer the larvae into a 50 milliliter tube. Return the filter paper to the dish and replace in the dark repeat collection of larvae on a weekly basis for up to one month. The collected larvae are then washed as described in the protocol text and stored at four degrees Celsius in distilled water.

To begin this procedure, soak the worms in approximately 10 milliliters of hang solutions, supplemented with 10%gentamicin for 20 minutes, leaving the tube resting at an angle to ensure worms are fully covered after 20 minutes. Wash the worms six times with Hank's solutions, supplemented with antibiotics, Eloqua worms into vented T 25 flasks. Place the flasks upright in a 37 degree Celsius incubator for three weeks, a preparation of excretory secretory products or HES from the worms.

HES containing culture. Media is collected from cultures at intervals of no longer than twice per week, replaced with an equal volume of h polyus Media on each occasion. Keep each collection separate and clearly labeled with the date and batch number due to greater potential for contamination with LPS and host proteins.

The first collection after 24 hours of culture should be set aside and either processed separately or discarded centrifuge HES containing media of 400 times G for five minutes. Then aspirate the media with a 50 milliliter syringe and pass through a 0.2 micron low protein binding filter into 50 milliliter tubes. Subsequently, the pooled HES Supena is concentrated over a 3000 molecular weight cutoff filter in an ultra filtration device under nitrogen pressure to set up the filter device first, wash the three kilo Dalton membrane shiny side down in a one liter beaker with distilled water while stirring.

Assemble the ultra filtration device per the manufacturer's instructions with the filter membrane shiny side up place in a cabinet of four degrees Celsius and pass 50 milliliters of distilled water through before starting to concentrate the poured HES. Be very careful not to let the filter run dry. Add each tube of HES into the filtration device as required until the volume is concentrated down to two to five milliliters in order to remove amino acids and other components of the tissue culture medium.

From the HES preparation, add 50 milliliters of pyro free PBS to the filtration device and then concentrate down to approximately two milliliters. Repeat this step twice. Using a total of 150 milliliters of PBS, transfer a ES into a syringe and filter sterilize with a 0.2 micron filter.

After measuring the protein concentration aliquot label with the batch number and date and freeze at minus 80 degrees Celsius. Oral gavage of 400 L three larvae is used to maintain the H poly gyus lifecycle in F1 mice. The resulting adult worm burdens are shown here.

Batches of HES have proven reproducible efficacy in functional assays and in protein composition. Moreover, when supernatants from each successive week of culture were analyzed, the protein profiles were found to be very similar for up to a total of four weeks. When a TS concentration is measured by the Bradford assay, the total protein is usually approximately one milligram per milliliter.

The yield of HES protein from 11 different batches derived from approximately 500 milliliter of culture s supernatant is shown. Avoidance of contamination is critical when collecting HES when levels of LPS contamination in 41 batches of HES were measured by the limbus AAMI CYTE assay. Most batches of HES were found to be significantly below the recommended contamination threshold.

While carrying out this procedure, it's important to remember to maintain careful sterile technique in setting up the cultures. Once mastered, this technique can be done in three to four hours if it's performed properly Following this procedure. Other methods like proteomics, protein fractionation, and analysis of glycans lipids and microRNAs can be performed in order to fully understand the spectrum of molecular products released from these parasites.

After watching this video, you should have a good understanding of how to propagate poly limus. Moid is polyus and collect DS products from the cultured adult worms for testing their effects on the immune system. While this parasite is not effective to humans, it is important to follow safety precautions and to adhere to the specified protocol to ensure reproducibility between batches of Hess.