Name: induction of drug-induced, autoimmune hepatitis in balb/c mice for the study of its pathogenic mechanisms
Uploaded: 2020-05-29T12:30:00
Description: Watch this Scientific Journal Video about Induction of Drug-Induced, Autoimmune Hepatitis in BALB/c Mice for the Study of Its Pathogenic Mechanisms at JoVE.com

Dr. Njoku would like to acknowledge Dr. Noel R. Rose, MD PhD, for his guidance and insightful discussions that resulted in the formulation of this model.

All procedures were approved by the animal care and use committee.
1. Trifluoroacetylation of hepatic S-100 cytosolic proteins or a CYP2E1 epitope
NOTE: First, prepare the trifluoroacetylated S100 (TFA-S100) and trifluoroacetylated CYP2E1 epitope (TFA-JHDN5). Because syngeneic S100 proteins are needed for immunizations, and BALB/c mice are required to produce the immunogen. The preparation yields a large amount of immunogen; so, anticipate performing this portion arou...

<ol>
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J., Perrone, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hapten+carrier+conjugates+associated+with+halothane+hepatitis.">Hapten carrier conjugates associated with halothane hepatitis.</a> Advances in Experimental Medicine and Biology. 283, 111-120 (1991).</li><li>Njoku, D. B., Talor, M. V., Fairweather, D., Frisancho-Kiss, S., Odumade, O. A., Rose, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+model+of+drug+hapten-induced+hepatitis+with+increased+mast+cells+in+the+BALB/c+mouse.">A novel model of drug hapten-induced hepatitis with increased mast cells in the BALB/c mouse.</a> Experimental and Molecular Pathology. 78 (2), 87-100 (2005).</li><li>Cottagiri, M., Nyandjo, M., Stephens, M., Mantilla, J., Saito, H., Mackay, I. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+drug-induced,+immune-mediated+hepatitis,+interleukin-33+reduces+hepatitis+and+improves+survival+independently+and+as+a+consequence+of+FoxP3++T-cell+activity.">In drug-induced, immune-mediated hepatitis, interleukin-33 reduces hepatitis and improves survival independently and as a consequence of FoxP3+ T-cell activity.</a> Cellular and Molecular Immunology. , (2018).</li><li>Lohse, A. W., Manns, M., Dienes, H. P., Meyer zum Buschenfelde, K. H., Cohen, I. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+autoimmune+hepatitis:+disease+induction,+time+course+and+T-cell+reactivity.">Experimental autoimmune hepatitis: disease induction, time course and T-cell reactivity.</a> Hepatology. 11 (1), 24-30 (1991).</li><li>McCarthy, E. K., Vakos, A., Cottagiri, M., Mantilla, J. J., Santhanam, L., Thomas, D. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+Shared+Cytochrome+p4502E1+Epitope+Found+in+Anesthetic+Drug-Induced+and+Viral+Hepatitis.">Identification of a Shared Cytochrome p4502E1 Epitope Found in Anesthetic Drug-Induced and Viral Hepatitis.</a> mSphere. 3 (5), (2018).</li><li>Cho, J., Kim, L., Li, Z., Rose, N. R., Talor, M. V., Njoku, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sex+bias+in+experimental+immune-mediated,+drug-induced+liver+injury+in+BALB/c+mice:+suggested+roles+for+Tregs,+estrogen,+and+IL-6.">Sex bias in experimental immune-mediated, drug-induced liver injury in BALB/c mice: suggested roles for Tregs, estrogen, and IL-6.</a> PLoS. One. 8 (4), 61186 (2013).</li><li>Satoh, H., Gillette, J. R., Takemura, T., Ferrans, V. J., Jelenich, S. E., Kenna, J. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigation+of+the+immunological+basis+of+halothane-induced+hepatotoxicity.">Investigation of the immunological basis of halothane-induced hepatotoxicity.</a> Advances in Experimental Medicine and Biology. 197, 657-673 (1986).</li><li>Eliasson, E., Kenna, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytochrome+P450+2E1+is+a+cell+surface+autoantigen+in+halothane+hepatitis.">Cytochrome P450 2E1 is a cell surface autoantigen in halothane hepatitis.</a> Molecular Pharmacology. 50 (3), 573-582 (1996).</li><li>Bourdi, M., Chen, W., Peter, R. M., Martin, J. L., Buters, J. T., Nelson, S. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+cytochrome+P450+2E1+is+a+major+autoantigen+associated+with+halothane+hepatitis.">Human cytochrome P450 2E1 is a major autoantigen associated with halothane hepatitis.</a> Chemical Research in Toxicology. 9 (7), 1159-1166 (1996).</li><li>Njoku, D. B., Li, Z., Washington, N. D., Mellerson, J. L., Talor, M. V., Sharma, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suppressive+and+pro-inflammatory+roles+for+IL-4+in+the+pathogenesis+of+experimental+drug-induced+liver+injury.">Suppressive and pro-inflammatory roles for IL-4 in the pathogenesis of experimental drug-induced liver injury.</a> European Journal of Immunology. 39 (6), 1652-1663 (2009).</li><li>Aithal, G. P., Ramsay, L., Daly, A. K., Sonchit, N., Leathart, J. 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V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sexual+dimorphism+in+autoimmunity.">Sexual dimorphism in autoimmunity.</a> Journal of Clinical Investigation. 125 (6), 2187-2193 (2015).</li><li>Satoh, H., Fukuda, Y., Anderson, D. K., Ferrans, V. J., Gillette, J. R., Pohl, L. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunological+studies+on+the+mechanism+of+halothane-induced+hepatotoxicity:+immunohistochemical+evidence+of+trifluoroacetylated+hepatocytes.">Immunological studies on the mechanism of halothane-induced hepatotoxicity: immunohistochemical evidence of trifluoroacetylated hepatocytes.</a> Journal of Pharmacology and Experimental Therapeutics. 233 (3), 857-862 (1985).</li><li>Habeeb, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+free+amino+groups+in+proteins+by+trinitrobenzenesulfonic+acid.">Determination of free amino groups in proteins by trinitrobenzenesulfonic acid.</a> Analytical Biochemistry. 14 (3), 328-336 (1966).</li><li>Christen, U., Burgin, M., Gut, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Halothane+metabolism:+immunochemical+evidence+for+molecular+mimicry+of+trifluoroacetylated+liver+protein+adducts+by+constitutive+polypeptides.">Halothane metabolism: immunochemical evidence for molecular mimicry of trifluoroacetylated liver protein adducts by constitutive polypeptides.</a> Molecular Pharmacology. 40 (3), 390-400 (1991).</li><li>Christen, U., Quinn, J., Yeaman, S. J., Kenna, J. G., Clarke, J. B., Gandolfi, A. 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The strength of this protocol rests in its reproducibility; so, it is critical to adhere to the suggested steps. Formulation of the immunogen can be a barrier for some; however, we have reproduced our model using the epitope described in our document, which removes the need to isolate the S100 fraction of the liver. It is likely that additional epitopes or proteins can be altered and induce hepatitis following immunizations; however, we describe those proteins that we have used with reliable results. Several proteins hav...

The purpose of this method is to describe a mouse model of drug-induced autoimmune hepatitis that develops in vivo and demonstrate how it can be utilized to investigate the molecular, immunologic and genetic basis of this disease. The long-term objective of our studies is to uncover mechanisms responsible for the development of chronic liver inflammation and injury by studying DIH in susceptible patients. Liver disease and cirrhosis constitute 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 hepatitis (DIH) is the third most common cause of acute liver failure in the United States. DIH is the most common hepatic drug-induced hypersensitization process observed in approximately 9 to 12% of patients with autoimmune hepatitis1. The overwhelming majority of patients with DIH are women2,3,4. A type of DIH develops in susceptible individuals following administration of halogenated volatile anesthetics such as isoflurane, sevoflurane, desflurane or halothane. These anesthetics covalently binds to liver proteins with reactive products of their metabolism, thus creating novel autoantigens capable of eliciting allergic or autoimmune responses5.
The study of pathogenic mechanisms involved in the development of anesthetic and any form of DIH has been previously hampered by the lack of an animal model that closely mimics the induction of human disease. We have developed an experimental murine model of DIH with features resembling immune-mediated DILI in patients. Hepatitis is induced by immunization with one of two autoantigens that have been covalently modified by the trifluoroacetyl chloride (TFA) metabolite that is formed following oxidative metabolism of the anesthetic by the enzyme cytochrome P450 2E1 (CYP2E1)5. One autoantigen is the hepatic cytosolic S100 liver fraction, which is a mixture of several proteins6, and the second autoantigen is an epitope of CYP2E1 that is recognized by sera from patients with anesthetic immune-mediated DILI7. By using BALB/c mice, which are relatively resistant to experimental autoimmune hepatitis, we distinguish our model from the S100-induced immunization model of autoimmune hepatitis in C57Bl/6J mice8.
Because of its diverse clinical presentations, DIH is difficult to study in patients. Translational experimental models offer the ability to evaluate the pathogenesis of disease in vivo and in vitro. At present, there are no other alternative methods for inducing DIH that fully examine in vivo or in vitro adaptive or innate immune responses without the use of animals. Moreover, since trifluoroacetylation of S-100 or the CYP2E1 epitope does not appear to produce an irritating immunogen, and we are inducing DIH by immunization with TFA-altered proteins, these animals will not receive ether, any halogenated anesthetic, barbiturate or alcohol prior to immunization or other procedures, considering that these agents may alter the parameters we are studying. Even so, we have decreased our mouse usage by utilizing computer simulation to confirm the binding preferences of our discovered CYP2E1 epitope9 and have mirrored human DIH implicating female sex by demonstrating that female BALB/c mice develop a more severe DIH10.
In spite of diverse presentations of DIH in patients and challenges in the study of clinical disease, post-translational modification of native proteins by reactive drug metabolites is an accepted key mechanism in the pathogenesis DIH that follows halogenated anesthetics11. Investigators also accept that CYP2E1 is a major autoantigen in this process12,13. The role of interleukin (IL)-4&#8211;upregulated CD4+T cells that recognize a post-translationally modified CYP2E1 and other liver proteins is an accepted initiator of anesthetic DIH by attracting neutrophils, eosinophils and mast cells into the liver14, and this mechanism has been confirmed in many forms of DIH15,16. Induced FoxP3-expressing CD4+CD25+T cells (Tregs) reduce the severity of DIH, and relative deficiencies of these cells in the spleen worsen DIH 10,7. Thus, the majority of advances in understanding DIH have been made possible by utilizing in vivo mouse models to evaluate the genetic, metabolic and immunologic mechanisms of DIH both in vivo and in vitro.
Because we and other investigators have uncovered roles for IL-4, neutrophils, and eosinophils in the initiation of DIH using different mouse models, we believe that this observation supports our contention that regardless of the DIH model utilized, hepatitis and injury are induced by IL-4. The strength of our protocol lies in the utilization of in vivo methodology, both male and female mice, and repetition of histology, CD4+ T cell proliferation assays and cytokines. The strength of our use of in vitro studies is that they reduce the numbers of mice needed while providing the methodology to isolate cellular interactions that drive DIH. We recommend the use of male and female mice because this reduces the possibility of unconscious bias in interpretation of results and strengthens the translation potential of our studies since the incidence, prevalence, and severity of DIH is higher in women17. We recommend that mice are obtained from a single vendor; however, if this is not possible, obtain litter mate controls or wild-type mice from the same vendor as the genetically altered mice.

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			<td height="42" style="height:42px;width:216px;">0.1% 2,4,6-trinitrobenzene sulfonic acid (TNBS)</td>
			<td style="width:71px;">ThermoFisher</td>
			<td align="right" style="width:119px;">28997</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">AKP Substrate Kit</td>
			<td style="width:71px;">BioRad</td>
			<td style="width:119px;">172-1063</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">BALB/c mice</td>
			<td style="width:71px;">Jackson</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">CellTrace&#8482; CFSE Cell Proliferation Kit</td>
			<td style="width:71px;">ThermoFisher</td>
			<td style="width:119px;">C34554</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">CFA H37Ra</td>
			<td style="width:71px;">Becton Dickinson (Difco Bacto)</td>
			<td align="right" style="width:119px;">231131</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FcR Blocking reagent</td>
			<td style="width:71px;">Milteyi</td>
			<td>130-092-575</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">General supplement</td>
			<td style="width:71px;">ThermoFisher</td>
			<td style="width:119px;">HPRG770</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">HepaRG&#8482; cells cryopreserved</td>
			<td style="width:71px;">ThermoFisher</td>
			<td style="width:119px;">HPR GC10</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Live/Dead Fixable Aqua Dead Cell stain kit&#160;</td>
			<td style="width:71px;">ThermoFisher</td>
			<td style="width:119px;">L34965</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">NaHC03</td>
			<td style="width:71px;">Millipore Sigma</td>
			<td style="width:119px;">S5761</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Percoll&#174;</td>
			<td style="width:71px;">Millipore Sigma</td>
			<td style="width:119px;">P1644-1L</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Pertussis Toxin</td>
			<td style="width:71px;">List Biologicals</td>
			<td align="right" style="width:119px;">180</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Phosphate Buffered Saline pH 7.4</td>
			<td style="width:71px;">Various</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Pierce&#8482; Protease Inhibitor Mini Tablets, EDTA Free</td>
			<td style="width:71px;">ThermoFisher</td>
			<td align="right" style="width:119px;">88666</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Potassium Hydroxide</td>
			<td style="width:71px;">JT Baker</td>
			<td style="width:119px;">3140-01</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">S-ethyltrifluorothioacetate (S-ETFA)</td>
			<td style="width:71px;">Millipore Sigma</td>
			<td align="right" style="width:119px;">177474</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Slide-a-lyzer dialysis cassettes (10 K, 12 ml)</td>
			<td style="width:71px;">ThermoFisher</td>
			<td align="right" style="width:119px;">66810</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">UltraPure&#8482; SDS Solution, 10%</td>
			<td style="width:71px;">ThermoFisher</td>
			<td align="right" style="width:119px;">24730020</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Williams Media E, no phenol red</td>
			<td style="width:71px;">ThermoFisher</td>
			<td style="width:119px;">A1217601</td>
			<td></td>
		</tr>
	</tbody>
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induction of drug-induced, autoimmune hepatitis in balb/c mice for the study of its pathogenic mechanisms

Drug-induced autoimmune hepatitis (DIH) is the most common hepatic drug-induced hypersensitization process observed in approximately 9 to 12% of patients with autoimmune hepatitis. The overwhelming majority of patients with DIH are women. The underlying mechanisms of these sex differences in prevalence are unclear because of the paucity of animal models that mimic human disease. Even so, underlying mechanisms are widely believed to be associated with human leukocyte antigen haplotypes and sex hormones. In contrast, using a DIH mouse model, we have uncovered that IL-4 initiated&#160;CD4+ T cells directed against an epitope of cytochrome P450 2E1 induces influx of neutrophils, macrophages and mast cells into the livers of female BALB/c mice. Using this model, we have also shown that IL-33-induced FoxP3+regulatory T cells confer protection against DIH in female and male mice. This DIH model is induced by immunizing mice with an epitope of CYP2E1 that has been covalently altered with a drug metabolite that has been associated with DIH. This epitope is recognized by patients with DIH. Our method induces robust and reproducible hepatitis and autoantibodies that can be utilized to study the pathogenesis of DIH. While in vivo studies can cause undue pain and distress in mice when done improperly, the advantage of an in vivo model is the ability to evaluate the pathogenesis of disease in a large number of mice. Additionally, biological effects of the altered liver proteins can be studied using invasive procedures. The addition of in vitro studies to the experimental design allows rapid repetition and mechanistic analysis at a cellular level. Thus, we will demonstrate our model protocol and how it can be utilized to study in vivo and in vitro mechanisms of DIH.

The immunization schedule utilized to induce DIH shown in Figure 1 represents the two immunizations required at the base of the neck (day 0) and the base of the tail (day 7). Figure 2 shows representative proliferation data obtained on day 14 using CFSE in response to CYP2E1, JHDN5, the CYP2E1 epitope and the trifluoroacetyl (TFA) metabolite of the anesthetics. Figure 3 shows the gating strategy and representative flow cytometry ana...

Watch this Scientific Journal Video about Induction of Drug-Induced, Autoimmune Hepatitis in BALB/c Mice for the Study of Its Pathogenic Mechanisms at JoVE.com

Induction of Drug-Induced, Autoimmune Hepatitis in BALB/c Mice for the Study of Its Pathogenic Mechanisms

We describe an in vivo immunization, translational hepatitis model in BALB/c mice that can be utilized to study the pathogenesis of drug-induced autoimmune hepatitis including sex differences seen in this disease. We will describe how this model demonstrates reproducible analyses using in vivo and in vitro experimental techniques.

Drug-induced autoimmune hepatitis (DIH) is the most common hepatic drug-induced hypersensitization process observed in approximately 9 to 12% of patients ...

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.

Research

JoVE Journal

Immunology and Infection

Preparation of Mouse Pituitary Immunogen for the Induction of Experimental Autoimmune Hypophysitis

Autoimmune hypophysitis is a chronic inflammation of the pituitary gland caused or accompanied by autoimmunity1.  It has traditionally been considered a ...

Induction of Experimental Autoimmune Hypophysitis in SJL Mice

Autoimmune hypophysitis can be reproduced experimentally by the injection of pituitary proteins mixed with an adjuvant into susceptible mice1. Mouse ...

Determination of Tolerable Fatty Acids and Cholera Toxin Concentrations Using Human Intestinal Epithelial Cells and BALB/c Mouse Macrophages

The positive role of fatty acids in the prevention and alleviation of non-human and  human diseases have been and continue to be extensively documented. ...

Myelin Oligodendrocyte Glycoprotein (MOG35-55) Induced Experimental Autoimmune Encephalomyelitis (EAE) in C57BL/6 Mice

Myelin Oligodendrocyte Glycoprotein MOG35-55 Induced Experimental Autoimmune Encephalomyelitis EAE in C57BL/6 Mice

Multiple sclerosis is a chronic neuroinflammatory demyelinating disorder of the central nervous system with a strong neurodegenerative component. While ...

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages ...

Immunofluorescence to Monitor the Cellular Uptake of Human Lactoferrin and its Associated Antiviral Activity Against the Hepatitis C Virus

Immunofluorescence is a laboratory technique commonly used to study many aspects of biology. It is typically used to visualize the distribution and/or ...

Induction of Experimental Autoimmune Encephalomyelitis in Mice and Evaluation of the Disease-dependent Distribution of Immune Cells in Various Tissues

Multiple sclerosis is presumed to be an inflammatory autoimmune disease, which is characterized by lesion formation in the central nervous system (CNS) ...

Development of a Hepatitis B Virus Reporter System to Monitor the Early Stages of the Replication Cycle

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), ...

Induction of Paralysis and Visual System Injury in Mice by T Cells Specific for Neuromyelitis Optica Autoantigen Aquaporin-4

While it is recognized that aquaporin-4 (AQP4)-specific T cells and antibodies participate in the pathogenesis of neuromyelitis optica (NMO), a human ...

Induction of Intestinal Graft-versus-host Disease and Its Mini-endoscopic Assessment in Live Mice

Acute graft-versus-host disease (GvHD) represents the most severe complication that patients previously undergoing allogeneic hematopoietic stem cell ...