This work was supported by NIH grant DK080351 to PC.

Experimental Protocol
The first step in this experimental protocol is to collect a large number of mouse pituitary glands. The second step is to homogenize the glands to prepare a protein extract. The third step is to emulsify these proteins with an oily mixture.
Step 1: Collection and storage of mouse pituitary glands
The mouse pituitary gland sits at the base of the skull in a depression of the sphenoid bone called the sella turcica, surrounded laterally by the trigeminal nerves and anteriorly by the optic chiasm. The gland is composed of a larger anterior lobe and smaller posterior and intermediate lobes, and weighs on average 1.9 mg. Pituitary glands are collected from mice of mixed strains, sexes, and ages, destined to be euthanized by the animal care facility.
To isolate the pituitary gland, the mouse is euthanized, the skin above the skull dissected, and the skull bone cut open with scissors. The brain is lifted to expose the pituitary gland, which is then isolated from the surrounding dura mater, scooped out of the sphenoid bone and stored in a plastic tube on dry ice.
In general we use 300 mouse pituitary glands for one protein preparation, a number that can be collected by one person in 9 hours.
Once the collection is completed, the tube containing the pituitary glands is stored at -80 &deg;C until ready for the next step.
Step 2. Extraction of mouse pituitary proteins
<ol>
<li>Add the homogenization buffer to a 15-ml Falcon tube (catalog no 352059) kept in an ice bath, using 250 &mu;L of buffer for every 100 pituitary glands collected. Use a buffer of ionic strength similar to the one that is physiologically present inside the cytoplasm of the cell (0.2 M) and of physiologic pH (7.4). We use phosphate buffered saline (0.161 M, pH 7.4), supplemented with a protease inhibitor cocktail (Sigma-Aldrich, catalog no P8340-1ML).</li>
<li>Add the pituitary glands to the buffer just before homogenization, and then place the generator (5-mm diameter) of the Polytron homogenizer at the bottom of the Falcon tube. Homogenize at maximum rpm for 30 seconds, gently moving the Falcon tube up and down while keeping it in the ice bath. Then, rest the homogenate on ice for 1 min. Repeat the homogenization and cooling steps two more times. This gentle homogenization should break the cells and release cytosolic proteins without disrupting the nuclei.</li>
<li>To remove nuclei, unbroken tissue and insoluble materials (such as connective tissue and denatured protein complexes), centrifuge the homogenate at low speed (1000g) for 10 min at 4 &deg;C.</li>
<li>Transfer the supernatant (now called Post-Nuclear Supernatant, PNS) to a new Falcon tube, taking care to leave the pellet undisturbed, and store the PNS on ice.</li>
<li>Resuspend the pellet in the original volume of homogenization buffer (250 &mu;L of buffer for every 100 pituitary glands collected), homogenize as above, centrifuge as above, and then combine the second PNS with the first one.</li>
<li>Aliquot the two pooled PNS samples into microcentrifuge tubes and store at -80 &deg;C until ready for the next step. This protein preparation can be stored at -80 &deg;C but gradually looses its potency, so we recommend to use it within 6 months from the preparation. Use a small aliquot to determine the protein yield and concentration by the BCA assay (Pierce catalog no. 23225), and the protein quality by gel electrophoresis. In general, we obtain about 60 mg of protein from 300 mouse pituitaries in 1.5 mL of homogenization buffer, at a concentration around 40 mg/ mL.</li>
</ol>
Step 3. Preparation of the emulsion for immunization
<ol>
<li>Thaw rapidly the frozen protein aliquots needed for the experiment and adjust the protein concentration to 20 mg/ mL. This protein extract will be emulsified 1:1 with Complete Freund's Adjuvant (CFA, Sigma F-5881) containing 5 mg/ mL of heat killed Mycobacterium tuberculosis (Becton Dickinson, 231141). Each mouse will receive 1 mg of pituitary extract in 100 &mu;L of emulsion.</li>
<li>The example given here is for immunizing 10 mice, where each mouse will be injected with 100 &mu;L of the emulsion containing 1 mg of pituitary proteins. Allow two extra mice for material loss during the preparation, so use 12 mg of pituitary proteins. Aspire 600 &mu;L of the 20 mg/ mL pituitary protein extract describe above using a 2.5 mL Hamilton gastight syringe. Resuspend the CFA by shaking, and aspire 600 &mu;L of CFA into another 2.5 mL Hamilton syringe. Attach and secure a 22-gauge micro-emulsifying needle to the syringe filled with CFA. Slowly push the plunger of the CFA syringe such that the needle is filled with CFA, then attach and secure the other end of the needle to the syringe filled with pituitary extract.</li>
<li>Slowly advance the plunger of the CFA syringe to push the oil component into the acqueous pituitary extract, such that only a small amount is mixed. Then push back the plunger of the pituitary extract syringe into the CFA syringe. Repeat this action gradually increasing the amount of material mixed. Continue until the entire content of the two syringes are mixed. You will obtain a whitish and evenly distributed solution that represents the water-in-oil emulsion.</li>
<li>Once the emulsion is formed, repeat the back-and-forth motion at least 500 times to ensure the emulsion is thoroughly mixed. After the final mixing cycle, the 1:1 water-in-oil emulsion is ready to use and contains a pituitary protein concentration of 10 mg/mL. The emulsion will be injected the same day subcutaneously into anesthetized SJL mice, as detailed in the companion JoVE article.</li>
</ol>

<ol>
	<li>Caturegli, P., Newschaffer, C., Olivi, A., Pomper, M. G., Burger, P. C., 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> Autoimmune Hypophysitis Endocr Rev. 26, 599-614 (2005).</li><li>Blansfield, J. A., Beck, K. E., Tran, K., Yang, J. C., Hughes, M. S., Kammula, U. S., Royal, R. E., Topalian, S. L., Haworth, L. R., Levy, C., Rosenberg, S. A., Sherry, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytotoxic+T-lymphocyte-associated+antigen-4+blockage+can+induce+autoimmune+hypophysitis+in+patients+with+metastatic+melanoma+and+renal+cancer.">Cytotoxic T-lymphocyte-associated antigen-4 blockage can induce autoimmune hypophysitis in patients with metastatic melanoma and renal cancer.</a> J Immunother. 28, 593-598 (2005).</li><li>Tzou, S. C., Lupi, I., Landek, M., Gutenberg, A., Tzou, Y. M., Kimura, H., Pinna, G., Rose, N. R., Caturegli, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autoimmune+Hypophysitis+of+SJL+mice:+Clinical+Insights+from+a+New+Animal+Model.">Autoimmune Hypophysitis of SJL mice: Clinical Insights from a New Animal Model.</a> Endocrinology. 149, 3461-3469 (2008).</li><li>Goudie, R. B., Pinkerton, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anterior+hypophysitis+and+Hashimoto's+disease+in+a+woman.">Anterior hypophysitis and Hashimoto's disease in a woman.</a> J Pathol Bacteriol. 83, 584-585 (1962).</li><li>Caturegli, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autoimmune+hypophysitis:+an+underestimated+disease+in+search+of+its+autoantigen(s).">Autoimmune hypophysitis: an underestimated disease in search of its autoantigen(s).</a> J Clin Endocrinol Metab. 92, 2038-2040 (2007).</li></ol>

No conflicts of interest declared.

Despite the fact that the first patient with autoimmune hypophysitis was reported in 19624, the pathogenic pituitary autoantigens remain to be identified5. Animal models can be very useful to identify these autoantigens, aiding the discovery of biomarkers that are translatable to patient care. This article has demonstrated a straightforward procedure for preparing pituitary proteins in a form that is adequate for inducing autoimmune hypophysitis in experimental animals.

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<td>15-ml Falcon tubes</td>
<td>&nbsp;</td>
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<td>homogenization buffer</td>
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<td>phosphate buffered saline</td>
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<td>protease inhibitor cocktail</td>
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<td>Polytron homogenizer</td>
<td>&nbsp;</td>
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<td>BCA assay</td>
<td>&nbsp;</td>
<td>Pierce</td>
<td>23225</td>
<td>&nbsp;</td>
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<td>Complete Freund's Adjuvant (CFA)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>F-5881</td>
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<td>Mycobacterium tuberculosis</td>
<td>&nbsp;</td>
<td>Becton Dickinson</td>
<td>231141</td>
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<td>2.5 mL Hamilton gastight syringe</td>
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<td>22-gauge micro-emulsifying needle</td>
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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 rare disease but reporting has increased markedly in recent years. Hypophysitis, in fact, develops not uncommonly as a "side effect" in cancer patients treated with antibodies that block inhibitory receptors expressed on T lymphocytes, such as CTLA-42 and PD-1 receptors. Autoimmune hypophysitis can be induced experimentally by injecting mice with pituitary proteins mixed with an adjuvant3. In this video article we demonstrate how to extract proteins from mouse pituitary glands and how to prepare them in a form suitable for inducing autoimmune hypophysitis in SJL mice.

Watch this Scientific Journal Video about Preparation of Mouse Pituitary Immunogen for the Induction of Experimental Autoimmune Hypophysitis at JoVE.com

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

Autoimmune hypophysitis can be reproduced in mice by injecting an extract of mouse pituitary proteins.

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

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Immunology and Infection

14.4K Views.  The Johns Hopkins University. Autoimmune hypophysitis can be reproduced in mice by injecting an extract of mouse pituitary proteins.

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

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 models allow us to study how diseases unfold, often providing a good replica of the same processes occurring in humans. For some autoimmune diseases, like type 1A diabetes, there are models (the NOD mouse) that spontaneously develop a disease similar to the human counterpart. For many other autoimmune diseases, however, the model needs to be induced experimentally. A common approach in this regard is to inject the mouse with a dominant antigen derived from the organ being studied. For example, investigators interested in autoimmune thyroiditis inject mice with thyroglobulin2, and those interested in myasthenia gravis inject them with the acetylcholine receptor3. If the autoantigen for a particular autoimmune disease is not known, investigators inject a crude protein extract from the organ targeted by the autoimmune reaction. For autoimmune hypophysitis, the pathogenic autoantigen(s) remain to be identified4, and thus a crude pituitary protein preparation is used. In this video article we demonstrate how to induce experimental autoimmune hypophysitis in SJL mice.

This video shows how to induce autoimmune hypophysitis in SJL mice and how to assess its severity by histopathology.

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Isolation of Infiltrating Leukocytes from Mouse Skin Using Enzymatic Digest and Gradient Separation

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Simple and Efficient Production and Purification of Mouse Myelin Oligodendrocyte Glycoprotein for Experimental Autoimmune Encephalomyelitis Studies

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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.

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Experimental Autoimmune Uveitis: An Intraocular Inflammatory Mouse Model

Experimental Autoimmune Uveitis (EAU) is driven by immune cells responding to self-antigens. Many features of this non-infectious, intraocular inflammatory disease model recapitulate the clinical phenotype of posterior uveitis affecting humans. EAU has been used reliably to study the efficacy of novel inflammatory therapeutics, their mode of action and to further investigate the mechanisms that underpin disease progression of intraocular disorders. Here, we provide a detailed protocol on EAU induction in the C57BL/6J mouse - the most widely used model organism with susceptibility to this disease. Clinical assessment of disease severity and progression will be demonstrated using fundoscopy, histological examination and fluorescein angiography. The induction procedure involves subcutaneous&#160;injection of an emulsion containing a peptide (IRBP1-20) from the ocular protein interphotoreceptor retinoid binding protein (also known as retinol binding protein 3), Complete Freund&#39;s Adjuvant (CFA) and supplemented with killed Mycobacterium tuberculosis. Injection of this viscous emulsion on the back of the neck is followed by a single intraperitoneal injection of Bordetella pertussis toxin. At the onset of symptoms (day 12-14) and under general anesthesia, fundoscopic images are taken to assess disease progression through clinical examination. These data can be directly compared with those at later timepoints and peak disease (day 20-22) with differences analyzed. At the same time, this protocol allows the investigator to assess potential differences in vessel permeability and damage using fluorescein angiography. EAU can be induced in other mouse strains - both wildtype or genetically modified - and combined with novel therapies offering flexibility for studying drug efficacy and/or disease mechanisms.

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A Rapid, Simple, and Standardized Homogenization Method to Prepare Antigen/Adjuvant Emulsions for Inducing Experimental Autoimmune Encephalomyelitis

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To induce experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis, mice are immunized with a water-in-oil emulsion containing an autoantigen and complete Freund's adjuvant. While several protocols exist for the preparation of these emulsions, a rapid, simple, and standardized homogenization protocol for emulsion preparation is presented here.

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Generation of a Mouse Spontaneous Autoimmune Thyroiditis Model

In recent years, Hashimoto's thyroiditis (HT) has become the most common autoimmune thyroid disease. It is characterized by lymphocyte infiltration and the detection of specific serum autoantibodies. Although the potential mechanism is still not clear, the risk of Hashimoto's thyroiditis is related to genetic and environmental factors. At present, there are several types of models of autoimmune thyroiditis, including experimental autoimmune thyroiditis (EAT) and spontaneous autoimmune thyroiditis (SAT).
EAT in mice is a common model for HT, which is immunized with lipopolysaccharide (LPS) combined with thyroglobulin (Tg) or supplemented with complete Freund's adjuvant (CFA). The EAT mouse model is widely established in many types of mice. However, the disease progression is more likely associated with the Tg antibody response, which may vary in different experiments.
SAT is also widely used in the study of HT in the NOD.H-2h4 mouse. The NOD.H2h4 mouse is a new strain obtained from the cross of the nonobese diabetic (NOD) mouse with the B10.A(4R), which is significantly induced for HT with or without feeding iodine. During the induction, the NOD.H-2h4 mouse has a high level of TgAb accompanied by lymphocyte infiltration in the thyroid follicular tissue. However, for this type of mouse model, there are few studies to comprehensively evaluate the pathological process during the induction of iodine.
A SAT mouse model for HT research is established in this study, and the pathologic changing process is evaluated after a long period of iodine induction. Through this model, researchers can better understand the pathological development of HT and screen new treatment methods for HT.

Several types of animal models of Hashimoto's thyroiditis have been established, as has spontaneous autoimmune thyroiditis in the NOD mouse. H-2h4 mice are a simple and reliable model for HT induction. This article describes this approach and evaluates the pathological process for a better understanding of the SAT murine model.

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Immunogenicity Enhancement by &lt;em&gt;Mycobacterium paratuberculosis&lt;/em&gt; in Autoimmune Encephalomyelitis

Adjuvant Activity of Mycobacterium paratuberculosis in Enhancing the Immunogenicity of Autoantigens During Experimental Autoimmune Encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) induced by myelin oligodendrocyte glycoprotein (MOG) requires immunization by a MOG peptide emulsified in complete Freund&#39;s adjuvant (CFA) containing inactivated Mycobacterium tuberculosis. The antigenic components of the mycobacterium activate dendritic cells to stimulate T-cells to produce cytokines that promote the Th1 response via toll-like receptors. Therefore, the amount and species of mycobacteria present during the antigenic challenge are directly related to the development of EAE. This methods paper presents an alternative protocol to induce EAE in C57BL/6 mice using a modified incomplete Freund&#39;s adjuvant containing the heat-killed Mycobacterium avium subspecies paratuberculosis strain K-10.
M. paratuberculosis, a member of the Mycobacterium avium complex, is the causative agent of Johne&#39;s disease in ruminants and has been identified as a risk factor for several human T-cell-mediated disorders, including multiple sclerosis. Overall, mice immunized with Mycobacterium paratuberculosis showed earlier onset and greater disease severity than mice immunized with CFA containing the strain of M. tuberculosis H37Ra at the same doses of 4 mg/mL. The antigenic determinants of Mycobacterium avium subspecies paratuberculosis (MAP)&#160;strain K-10 were able to induce a strong Th1 cellular response during the effector phase, characterized by significantly higher numbers of T-lymphocytes (CD4+ CD27+), dendritic cells (CD11c+ I-A/I-E+), and monocytes (CD11b+ CD115+) in the spleen compared to mice immunized with CFA. Furthermore, the proliferative T-cell response to the MOG peptide appeared to be highest in M. paratuberculosis-immunized mice. The use of an encephalitogen (e.g., MOG35-55) emulsified in an adjuvant containing M. paratuberculosis in the formulation may be an alternative and validated method to activate dendritic cells for priming myelin epitope-specific CD4+ T-cells during the induction phase of EAE.

Author Spotlight: Adjuvant Activity of Mycobacterium paratuberculosis in Enhancing the Immunogenicity of Autoantigens During Experimental Autoimmune Encephalomyelitis  

Here, we present an alternative protocol to actively induce experimental autoimmune encephalomyelitis in C57BL/6 mice, using the immunogenic epitope myelin oligodendrocyte glycoprotein (MOG)35-55 suspended in incomplete Freund's adjuvant containing the heat-killed Mycobacterium avium subspecies paratuberculosis.

Experimental autoimmune encephalomyelitis (EAE) induced by myelin oligodendrocyte glycoprotein (MOG) requires immunization by a MOG peptide emulsified in ...

Investigating Humoral Response in Autoimmune Demyelination

Quantification of Autoreactive Antibodies in Mice upon Experimental Autoimmune Encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is a disease model that recapitulates the autoimmune disorder multiple sclerosis (MS) at histopathological and molecular levels. EAE is induced by immunizing experimental animals via subcutaneous injection of short myelin peptides together with specific adjuvants to boost the immune response. Like the human counterpart, EAE mice develop demyelinating lesions, immune cell infiltration into the central nervous system (CNS), glia activation and neuronal injury. A consistent body of evidence also supports a mechanistic role for B cell dysfunction in the etiology of both MS and EAE. B cells can serve as antigen-presenting cells as well as a primary source of pro-inflammatory cytokines and autoantibodies. In EAE, antibodies are generated against the myelin peptides that were employed to induce the disease. Such autoantibodies have been shown to mediate either myelin loss or pathogenic T cell reactivation into the CNS. This article describes an efficient ELISA-based protocol to quantify autoantibodies in the serum of C57BL/6J mice immunized with the myelin oligodendrocyte glycoprotein 35-55 (MOG35-55) peptide. The proposed method serves as a powerful tool to investigate the specificity and magnitude of the aberrant humoral response in the context of autoimmune demyelination.

Author Spotlight: Novel Assay for Studying B-Cell Responses in Multiple Sclerosis Research

Experimental autoimmune encephalomyelitis (EAE) is an animal model of multiple sclerosis (MS), which shares with the human disease a robust humoral autoimmune response. Here, we report a simple and flexible ELISA protocol to quantify autoantibodies in the serum of EAE immunized mice.

Experimental autoimmune encephalomyelitis (EAE) is a disease model that recapitulates the autoimmune disorder multiple sclerosis (MS) at histopathological ...