Liver disease and cirrhosis are the sixth most common cause of death in adults between the ages of 25 and 64. Idiosyncratic DILI, sometimes referred to as drug-induced, autoimmune, or immune-mediated hepatitis, is the third most common cause of acute liver failure in the United States, and is the most common hepatic drug-induced hyposensitization process. This process occurs in about 9 to 12%of patients with autoimmune hepatitis, and this form of hepatitis is the most common reason that a drug is removed from the commercial market.
The overwhelming majority of patients with drug-induced hepatitis are women. This protocol describes critical features seen in patients such as hepatitis, auto-antibodies, and sex differences. The main advantage of this technique is its reproducibility in an in vivo mouse model, which gives scientists a chance to watch the development of disease and its sequela.
Using this protocol, we discovered a common epitope in anesthetic and viral hepatitis that is a dominant epitope in patients with anesthetic hepatitis, and is significantly associated with fibrosis in patients with viral hepatitis. We believe that this information can be used to develop specific diagnostic tests for drug-induced or viral hepatitis or associated fibrosis that hopefully can predict individuals at risk for developing this disease or their sequela. The written techniques can be overwhelming because we always use several checks and balances to ensure reproducibility of our results.
The first time you do the experiment, give yourself enough time. After the first time through, it goes pretty quickly. After euthanasia of the BALB/c mouse, use a midline incision to expose the intraabdominal contents with microsurgical scissors and make a small cut in the inferior vena cava to remove the blood.
Then place a 24-gauge angiocatheter into the portal vein. Keep the PBS in a water bath at four degrees Celsius and use a peristaltic pump to profuse the liver with 40 milliliters of PBS at the rate of 10 milliliters per minute. With microscissors, carefully release the liver from the abdomen and weigh the liver on a tared weighing boat.
Cut the liver into small 10-to 15-millimeter pieces. Add the liver samples as well as the homogenization buffer, four times the weight of a mouse liver, to a 15-milliliter polypropylene tube and supplement with complete protease inhibitor cocktail tablets according to the manuscript. Use a general laboratory tissue homogenizer on medium speed to homogenize the samples on ice until smooth.
Centrifuge the liver homogenates at 1, 500 times G for 10 minutes and then pour off the supernatant cytosolic S100 After centrifugation again at 100, 000 times G, snap-freeze the supernatant cytosolic S100 in dry ice and freeze the tube at minus 80 degrees Celsius. To begin trifluoroacetylation, thaw the S100 at room temperature. Then, in an Erlenmeyer flask, dilute 20 milligrams of mouse S100 to 10 milliliters with deionized water.
With the pH meter, adjust the pH to 10 with 1-normal potassium hydroxide. In a fume hood, add 4.7 millimoles of S-ETFA to the solution. Maintain the pH between 9.9 to 10.0 by administering 1-normal potassium hydroxide in droplet fashion for approximately one hour.
Record the total volume of potassium hydroxide for each reaction. Transfer the solutions into separate dialysis cassettes. Place the cassettes in four liters of deionized water to dialyze for 72 hours with three changes per day.
After dialysis, record the final volume of trifluoroacetylated S100 and then aliquot into labeled tubes. Snap-freeze the tubes in dry ice and store at minus 80 degrees Celsius. With the protein concentration determined according to the manuscript, if it is greater than one milligram per milliliter, add one milligram of each native and TFA-altered protein to one milliliter deionized water in separate bullet tubes.
Prepare a blank in the bullet tube using one milliliter deionized water. In a 96-well plate, add 50 microliters of the blank, native, and altered proteins each, in duplicate. Then, add 50 microliters of 4%sodium bicarbonate solution followed by 50 microliters of 0.1%2, 4, 6-trinitrobenzene sulfonic acid to each well.
Incubate the plate at 40 degrees Celsius for two hours. Following the incubation, add 50 microliters of 10%SDS to each well, followed by 25 microliters of 1-normal hydrochloric acid. After immunization and anesthetization of the mouse, use microsurgical scissors to expose the intraabdominal cavity.
Identify the spleen. Cut the spleen at the pedicle and place it in a Petri dish with PBS containing 2%fetal calf serum. To release the cells, wedge the spleen between two frosted glass slides and rub them together.
Use an electronic pipette to transfer the cells to a 50-milliliter conical polypropylene tube. To wash, add PBS containing 2%FCS to the tube to reach the volume of 50 milliliters and centrifuge at 335 times G using a benchtop refrigerated centrifuge. Pour off the supernatant and repeat the washing step.
Add one milliliter of ACK lysing buffer into the tube to wash for one minute for removing red cells. Then repeat the FCS-supplemented PBS washing step. After labeling cells with CFSE for 30 minutes, add 10 micrograms per milliliter of cytochrome-P450 2E1, JHDN5, or trifluoroacetylated ovalbumin to stimulate labeled cells for 72 hours at 37 degrees Celsius in the incubator.
After incubation, stain the cells with CD4 labeled with allophycocyanin at a ratio of 1:100 for 30 minutes on ice. After laparotomy of the immunized mouse as described in the manuscript, cannulate the portal vein with a 25-gauge needle and then cut the inferior vena cava below the renal veins. In a 37-degree-Celsius water bath, perfuse each liver with 40 milliliters of PBS at a flow rate of 10 milliliters per minute.
After perfusion, using microsurgical scissors, cut the liver at the hepatic pedicle, remove the gallbladder, and then cut the liver at the hilum. On a 300-mesh stainless steel sieve, use a 20-milliliter sterile syringe pestle and cold PBS to disrupt the liver. With the screen, filter the resulting cell suspension into 50-milliliter pre-sterilized centrifuge tubes.
Bring each suspension to 50 milliliters using cold PBS and then wash the suspension by centrifugation for 10 minutes at 370 times G Discard the supernatant and then combine pellets by treatment into new 50-milliliter tubes. Suspend the combined pellets in 45 milliliters of 35%Percoll in PBS and 100 international units per milliliter heparin. Then, spin each tube at 500 times G for 10 minutes at 20 degrees Celsius.
Discard the supernatant and suspend the pellet in five milliliters of PBS and then add one milliliter of ACK lysing buffer to each pellet and store on ice for 10 minutes. After washing again with PBS by centrifugation at 370 times G, discard the supernatant and then wash the cells with FCS-supplemented PBS by centrifugation for 10 minutes at 370 times G.Use an electronic hemocytometer to count the cells. In this experiment, the CD4+T cell immune responses were determined using flow cytometry.
Representative proliferation data was obtained on day 14 using CFSE in response to trifluoroacetyl metabolite, cytochrome-P450 2E1, the cytochrome-P450 2E1 epitope, JHDN5. Wells without antigen were used as controls. When compared to the control, BALB/c mice developed proliferation in response to trifluoroacetylated ovalbumin and cytochrome-P450 2E1, but not JHDN5.
Three weeks after TFA emulsified in complete Freund's adjuvant immunization, female BALB/c mice developed more drug-induced autoimmune hepatitis when compared to male BALB/c mice. Histological analysis of liver tissues also shows more severe hepatitis inflammation in female mice than in male mice. Numbers of hepatic CD4+CD8+NK+and NKT+cells were significantly higher in females than in males.
while there were no differences in B cells seen between these mice Remember that although it is a long day, this formulation will last a long time. This procedure can also be performed to trifluoroacetylate the epitope. This procedure allowed researchers to dissect the time course of liver disease development and increase the potential for new discoveries of proteins and pathways.
S-ethyltrifluorothioacetate is toxic, and should be handled in a fume hood. Additionally, good lab practice and techniques are strongly recommended.
