This work was supported in part by the National Institutes of Health grants DK125011, AI150210, and DK124132, the University of Texas System STARs award (Y.C.), and the James W. McLaughlin Fellowship Fund from The University of Texas Medical Branch at Galveston (W.Y.). Figure 1 was created with BioRender.com.

All animal procedures were performed according to the University of Texas Medical Branch&#39;s Committee on the Use and Care of the animals. CBir1 TCR Tg mice were provided by Dr. Charles Elson of the University of Alabama at Birmingham. CBir1 TCR Tg mice can be female or male but should be at 8-12 weeks. Rag1-/- mice on the C57BL/6 background were obtained from the Jackson Laboratory10. Rag1-/- mice must be gender and age-matched, and either male or female can be used but should be at 8-12 weeks. The entire protocol is summarized in Figure 1.
1. Preparation of the recipient mice
<ol>
	<li>Prepare Rag1-/- mice on the C57BL/6 background, bred in the same specific pathogen-free animal facility. Calculate the number of mice per group by power analysis11. 
	NOTE: Rag1-/- mice do not have mature T cells and B cells10.</li>
	<li>Mark the mice by ear punch.</li>
	<li>Weigh the mice on the same day of T cell transfer.</li>
</ol>
2. Preparation of the reagents and solutions
NOTE: The reagents used are toxic, or biohazard and their handling need precautions and safety measures.
<ol>
	<li>Prepare Washing Buffer: add 5 mL of 100x Penicillin-Streptomycin into 500 mL of RPMI 1640 medium. Mix it thoroughly and store it at 4 &#176;C.</li>
	<li>Prepare Tris-NH4Cl Lysis Buffer.
	<ol>
		<li>Prepare Solution Part A. Dissolve 2.06 g of Tris base in 100 mL of double-distilled water (ddH2O) and adjust the pH value to 7.2 with HCl.</li>
		<li>Prepare Solution Part B. Dissolve 7.47 g of NH4Cl in 800 mL of ddH2O.</li>
		<li>Mix A and B thoroughly. Measure the pH and adjust it to 7.2 if not.</li>
		<li>Adjust the total volume to 1000 mL. Autoclave and then store it at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Prepare the Isolation Buffer.
	<ol>
		<li>Add 2.5 g of BSA and 500 &#181;L of EDTA (0.5 M, pH 8.0) into 500 mL of 1x PBS. Mix it thoroughly.</li>
		<li>Filter the solution through a 0.22 &#181;m vacuum-driven disposable bottle top filter (see Table of Materials). Store it at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Prepare FACS Buffer. Add 1 mL of FBS and 50 &#181;L of EDTA (0.5 M, pH 8.0) into 50 mL of Washing Buffer (prepared in step 2.1). Mix thoroughly and store it at 4 &#176;C.</li>
	<li>Prepare Complete Medium. Add 5 mL of FBS in 45 mL of Washing Buffer. Mix thoroughly and store it at 4 &#176;C.</li>
	<li>Prepare EDTA-PBS Buffer.
	<ol>
		<li>Calculate the volume of the EDTA-PBS Buffer needed. Volume (mL) = mouse number x 20.</li>
		<li>Add appropriate volume of FBS, EDTA, and HEPES in the PBS (2% of FBS, 0.5 mM of EDTA, 10 mM of HEPES in PBS) (see Table of Materials).</li>
		<li>Mix it thoroughly and pre-warm in a 37 &#176;C water bath.</li>
	</ol>
	</li>
	<li>Prepare the Digestion Buffer.
	<ol>
		<li>Calculate the volume of the Digestion Buffer needed. Volume (mL) = mouse number x 10.</li>
		<li>Add appropriate volume of FBS, Collagenase IV, and DNase I in Washing Buffer (2% of FBS, 0.5 mg/mL of Collagenase IV, and 10 U/mL of DNase I in Washing Buffer) (see Table of Materials).</li>
		<li>Mix it thoroughly and pre-warm in a 37 &#176;C water bath.</li>
	</ol>
	</li>
	<li>Prepare Percoll Solution.
	<ol>
		<li>Prepare 100% Percoll. Add 5 mL of 10x PBS in 45 mL of original Percoll (see Table of Materials).</li>
		<li>Prepare 2% FBS in Washing Buffer. Add 1 mL of FBS in 49 mL of Washing Buffer.</li>
		<li>Caulculate the volume of 40 % Percoll Solution and 75 % Percoll Solution. Volume of 40% Percoll Solution (mL) = mouse number x 4; Volume of 75% Percoll Solution (mL) = mouse number x 2. 
		NOTE: The reagents/solutions prepared in steps 2.1-2.5 will be used in steps 3-4, and those prepared in steps 2.6-2.8 will be used in step 9. All the reagents/solutions used in step 9 should be freshly prepared. Making 5 % extra Buffer is recommended for all the steps.</li>
	</ol>
	</li>
</ol>
3. Isolation of splenic CBir1 TCR Tg CD4+ T cells
<ol>
	<li>Euthanize CBir1 TCR Tg mouse/mice by a cervical dislocation with CO2 euthanasia (30%-70% gas-air displacement rate). Wet the mice with 70% ethanol.</li>
	<li>Perform a ~1 cm left abdomen incision, pull the skin away from the abdominal muscle tissue, make a ~3 cm incision in the abdominal muscle tissue, and remove the spleen with sterile scissors and forceps. Place the spleen in a culture dish containing 5 mL of pre-cold Washing Buffer (prepared in step 2.1).</li>
	<li>Grind the spleen with the rough surface of two sterile glass slides. Transfer the cell suspension into a 50 mL centrifuge tube by passing through a 100 &#181;m cell strainer (see Table of Materials). Rinse the glass slides and culture dish with 5 mL of pre-cold Washing Buffer and transfer the Washing Buffer into the tube.</li>
	<li>Centrifuge the cell suspension at 350 x g for 8 min at 4 &#176;C. Discard the supernatant and resuspend the cells with 5 mL of pre-warmed Tris-NH4Cl Lysis Buffer (prepared in step 2.2) per spleen. Incubate for 10 min at room temperature. Add 10 mL of pre-cold Washing Buffer to the tube.</li>
	<li>Centrifuge the cell suspension at 350 x g for 8 min at 4 &#176;C. Discard the supernatant and resuspend the cells with 10 mL of pre-cold Isolation Buffer (prepared in step 2.3).</li>
	<li>Count the cells. Mix 10 &#181;L of cell suspension with 10 &#181;L of trypan blue thoroughly. Load 10 &#181;L of the mixture onto a slide, insert the slide into the Automated Cell Counter (see Table of Materials) and obtain the viable cell number12. 
	NOTE: Approximately 1 x 108 cells can be obtained from one donor mouse in this step.</li>
	<li>Centrifuge the remaining cell suspension from step 3.6 at 350 x g for 8 min at 4 &#176;C. Discard all the supernatants.</li>
	<li>Vortex the anti-mouse CD4 Magnetic Particles thoroughly (see Table of Materials), directly add 50 &#181;L of the particles per 107 cells and mix with cell pellets thoroughly. Incubate for 30 min at 4 &#176;C. 
	NOTE: Any other commercial CD4+ T cell enrichment kit can be used here.</li>
	<li>Transfer the cell-particle suspension into a sterile collection tube. Add 3.5 mL of pre-cold Isolation Buffer into the tube.</li>
	<li>Place the tube on the Cell Separation Magnet (see Table of Materials) for 8 min at room temperature. Carefully aspirate off the supernatant using a 3 mL Transfer Pipette.</li>
	<li>Remove the tube from Cell Separation Magnet (see Table of Materials), resuspend the cells with 3.5 mL pre-cold Isolation Buffer, and place the tube to the Magnet for 4 min at room temperature. Carefully aspirate off the supernatant using a 3 mL Transfer Pipette.</li>
	<li>Repeat step 3.11.</li>
	<li>Resuspend the cells with 1 mL of pre-cold FACS Buffer (prepared in step 2.4).</li>
</ol>
4. Purification of CBir1 TCR Tg na&#970;ve CD4+ T cells
<ol>
	<li>Count the cells following step 3.6.</li>
	<li>If the cell concentration is &#62;107/mL, add a volume of FACS buffer to make sure the cell concentration is &#8804; 107/mL. 
	NOTE: ~1 &#215; 107 cells can be obtained from one donor mouse in this step.</li>
	<li>Stain the surface markers with 10 &#181;L of anti-mouse CD4-APC, 10 &#181;L of anti-mouse CD25-Percp/Cy5.5, and 10 &#181;L of anti-mouse CD62L-PE13,14 (see Table of Materials). Mix gently and incubate for 30 min at 4 &#176;C in the dark.</li>
	<li>Wash the cells with 2 mL of pre-cold FACS buffer. Centrifuge the cell suspension at 350 x g for 8 min at 4 &#176;C. Aspirate all the supernatantsusing a 3 mL Transfer Pipette.</li>
	<li>Repeat Step 4.4.</li>
	<li>Resuspend the cells to the concentration of 40 x 106/mL in pre-cold FACS buffer. 
	NOTE: To prevent the sorter from clogging, pass the cells through a 70 &#181;m strainer.</li>
	<li>Add 0.1 &#181;g/mL of DAPI. 
	NOTE: DAPI is used for excluding the dead cells.</li>
	<li>Prepare 15 mL centrifuge tubes containing 4 mL of Complete Medium (prepared in step 2.5) for collecting the sorted cells.</li>
	<li>Load the cells onto the sorter. Sort single viable na&#970;ve CD4+ T cells (DAPI- CD4+ CD25- CD62L+ cells) in purity mode (Nozzle size: 70 &#181;m; Pressure: 70 PSI; Event rate: 8000-12000 events/s; Efficiency: higher than 90%) (Figure 2). 
	NOTE: Na&#970;ve CD4+ T cells express high expression of CD62L and lack the activation marker CD2513,14.</li>
	<li>Centrifuge the cell suspension at 350 x g for 8 min at 4 &#176;C. Aspirate all the supernatants using a 3 mL Transfer Pipette.</li>
	<li>Resuspend the cells in 500 &#181;L of 1x PBS.</li>
	<li>Count cells following step 3.6 
	NOTE: ~5 &#215; 106 cells can be obtained from one donor mouse in this step.</li>
</ol>
5. Cell transfer into the recipient mice
<ol>
	<li>Resuspend the CBir1 TCR Tg na&#970;ve CD4+ T cells to the 5 x 106/mL concentration by adding 1x PBS.</li>
	<li>Warm the Rag-/- mice under a heat lamp (see Table of Materials) for 4 min, and restrain the mice using a mouse restrainer.</li>
	<li>Intravenously transfer 200 &#181;L of the cell suspension into the tail vein of Rag-/- mice using a 1 mL insulin syringe (27 G) (see Table of Materials). 
	NOTE: Cells from one donor mouse are enough to transfer to around five recipient mice.</li>
</ol>
6. Monitoring clinical signs during colitis progression
<ol>
	<li>Weigh the mice every week and increase the observation to twice a week once the mice start losing &#62;5% of original weights.</li>
	<li>Observe mice response/move when gently stimulated.</li>
	<li>Observe other clinical abnormalities. i.e., posture and stool consistency.</li>
</ol>
7. Colon collection and histopathological scoring
<ol>
	<li>Sacrifice the recipient mice by a cervical dislocation with CO2 at a time point of the weight loss &#62;20% of original weight or 6-weeks post cell transfer. Wet the mice with 70% ethanol.</li>
	<li>Perform a ~1 cm ventral midline skin incision, pull the skin away from the abdominal muscle tissue, make a ~3 cm incision in the abdominal muscle tissue, identify the cecum, and remove the entire colon with sterile scissors and forceps. Wet the colon with pre-cold PBS in a culture dish.</li>
	<li>Incise the colon lengthwise and rinse it with pre-cold PBS. Cut 1/3 of the colon longitudinally.</li>
	<li>Place the colon strip in a paper towel with the luminal side facing upward. Perform Swiss rolling using a toothpick15.</li>
	<li>Place the colon Swiss into a cassette and put the cassette in 10% buffered formalin for 24 h16, followed by dehydration&#160;using an automated processor (see Table of Materials) and paraffin embedding.</li>
	<li>Cut 5 &#181;m tissue sections on a microtome, mounted on slides, and perform the Hematoxylin and eosin (H&#38;E) stain17 (see Table of Materials).</li>
	<li>Determine the histopathological scores by combining the scores for each of the six parameters for a maximum of 12. Lamina propria inflammation (normal, 0; mild, 1; moderate, 2; severe, 3); goblet cell loss (normal, 0; mild, 1; moderate, 2; severe, 3); abnormal crypt (normal, 0; hyperplastic, 1; disorganization, 2; crypt loss, 3); crypt abscesses (absent, 0; present, 1); mucosal erosion and ulceration (normal, 0; mild, 1; moderate, 2; severe, 3); and submucosal change (none, 0; submucosa, 1; transmural, 2)18.</li>
</ol>
8. Isolation and staining of intestinal lamina propria cells
<ol>
	<li>After step 7.3, cut another 2/3 of the colon into 0.5-1 cm pieces and wash it with pre-cold PBS.</li>
	<li>Transfer the colon segments into 20 mL pre-warmed EDTA-PBS buffer in a 50 mL centrifuge tube. Incubate at 37 &#176;C with 250 rpm shaking for 30 min.</li>
	<li>Vortex the tube, discard the supernatants by passing it through a sterile sieve (diameter: 0.01 inches), and resuspend the colon segments in 20 mL of pre-cold PBS in the 50 mL tube.</li>
	<li>Repeat step 8.3 twice.</li>
	<li>Place the colon segments in a C tube (see Table of Materials) containing 10 mL of pre-warmed Digestion Buffer.</li>
	<li>Place the tube on a Dissociator machine (see Table of Materials) and incubate under the program of &#34;37C_m_LPDK_1&#34; for 25 min. 
	NOTE: &#34;37C_m_LPDK_1&#34; is a standard preset program in the Dissociator machine used for stirring the samples and keeping them at 37 &#176;C.</li>
	<li>Check if tissue is digested completely, which means that no piece of tissue is in the Digestion buffer. If not, repeat the program of &#34;37C_m_LPDK_1&#34;.</li>
	<li>Collect the supernatant by passing through a metal sieve and 100 &#181;L strainer. Rinse with 10 mL of pre-cold PBS.</li>
	<li>Centrifuge the cell suspension at 350 x g for 8 min at 4 &#176;C. Aspirate all the supernatants.</li>
	<li>Resuspend the cells in 4 mL of 40% Percoll Solution and mix it thoroughly (prepared in step 2.8).</li>
	<li>Transfer the resuspended cells to 2 mL of 75% Percoll solution in a 15 mL centrifuge tube.</li>
	<li>Centrifuge the cell suspension at 850 x g for 20 min at 20 &#176;C (Acceleration ramp: 0; Brake ramp: 0).</li>
	<li>Carefully remove fat on the top layer using a 3 mL Transfer Pipette and transfer the cell layer to 20 mL of Washing Buffer in a 50 mL tube.</li>
	<li>Centrifuge the cell suspension at 350 x g for 8 min at 4 &#176;C. Discard all the supernatants and resuspend cells in 1 mL of Completed Medium.</li>
	<li>Count the cells following step 3.6.</li>
	<li>Seed the cells in a 24-well plate, activate them with 50 ng/mL of Phorbol-12-myristate 13-acetate and 750 ng/mL of ionomycin for 2 h, followed by incubation with 5 &#181;g/mL of Brefeldin A for 3 h (see Table of Materials). 
	NOTE: The reagents used are toxic, and their handling needs precautions and safety measures.</li>
	<li>Transfer the cells into a FACS tube, add 2 mL of FACS Buffer, and centrifuge the cells at 350 x g for 8 min at 4 &#176;C. Discard the supernatant.</li>
	<li>Incubate the cells with 12.5 &#181;g/mL of anti-mouse CD16/3219 in (see Table of Materials) FACS buffer to block Fc receptors for 5 min at room temperature.</li>
	<li>Stain for live/dead and surface marker.
	<ol>
		<li>Wash the cells with 2 mL of FACS Buffer and centrifuge them at 350 x g for 8 min at 4 &#176;C. Discard the supernatant.</li>
		<li>Stain the cells with live dye and surface markers (i.e.,anti-mouse CD3 and anti-mouse CD4 antibodies)20 (see Table of Materials) in FACS Buffer at the optimized concentration for 30 min at 4 &#176;C in the dark. 
		NOTE: The reagents used are toxic, and their handling needs precautions and safety measures.</li>
	</ol>
	</li>
	<li>Perform the cellular and nuclear staining.
	<ol>
		<li>Add 2 mL of FACS Buffer and centrifuge the cell suspension at 350 x g for 8 min at 4 &#176;C. Discard the supernatant.</li>
		<li>Permeabilize and fix the cells by resuspending the cells with 200 &#181;L of Transcription Factor Fix working solution (see Table of Materials) for 40 min at room temperature.</li>
		<li>Add 2 mL of Perm buffer and centrifuge the cell suspension at 350 x g for 8 min at 4 &#176;C. Discard the supernatant.</li>
		<li>Incubate the cells with cellular and nuclear markers (i.e., anti-mouse IFN&#947;, anti-mouse IL-17A, and anti-mouse Foxp3 antibodies)20 in Perm buffer (see Table of Materials) for 30 min-1 h at room temperature.</li>
		<li>Add 2 mL of Perm buffer and centrifuge the cell suspension at 350 x g for 8 min at 4 &#176;C. Discard the supernatant.</li>
		<li>Add 1 mL of FACS Buffer and centrifuge the cell suspension at 350 x g for 8 min at 4 &#176;C. Discard the supernatant.</li>
	</ol>
	</li>
	<li>Resuspend cells with 200 &#181;L of FACS Buffer and run the samples on a flow cytometer (see Table of Materials).</li>
</ol>

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Role of the Intestinal Microbiota.</a> Inflammatory Bowel Disease. 24 (2), 361-379 (2018).</li><li>Bamias, 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=Down-regulation+of+intestinal+lymphocyte+activation+and+Th1+cytokine+production+by+antibiotic+therapy+in+a+murine+model+of+Crohn's+disease.">Down-regulation of intestinal lymphocyte activation and Th1 cytokine production by antibiotic therapy in a murine model of Crohn's disease.</a> Journal of Immunology. 169 (9), 5308-5314 (2002).</li><li>Steinbach, E. C., Gipson, G. R., Sheikh, S. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+murine+intestinal+inflammation+by+adoptive+transfer+of+effector+CD4++CD45RB+high+T+cells+into+immunodeficient+mice.">Induction of murine intestinal inflammation by adoptive transfer of effector CD4+ CD45RB high T cells into immunodeficient mice.</a> Journal of Visualized Experiments. (98), e52533 (2015).</li><li>Atale, N., Gupta, S., Yadav, U. C., Rani, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-death+assessment+by+fluorescent+and+nonfluorescent+cytosolic+and+nuclear+staining+techniques.">Cell-death assessment by fluorescent and nonfluorescent cytosolic and nuclear staining techniques.</a> Journal of Microscopy. 255 (1), 7-19 (2014).</li><li>Manichanh, C., Borruel, N., Casellas, F., Guarner, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+gut+microbiota+in+IBD.">The gut microbiota in IBD.</a> Nature Reviews Gastroenterology &amp; Hepatology. 9 (10), 599-608 (2012).</li><li>Sun, M., 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=Microbiota-derived+short-chain+fatty+acids+promote+Th1+cell+IL-10+production+to+maintain+intestinal+homeostasis.">Microbiota-derived short-chain fatty acids promote Th1 cell IL-10 production to maintain intestinal homeostasis.</a> Nature Communications. 9 (1), 3555 (2018).</li><li>Feng, T., 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=Th17+cells+induce+colitis+and+promote+Th1+cell+responses+through+IL-17+induction+of+innate+IL-12+and+IL-23+production.">Th17 cells induce colitis and promote Th1 cell responses through IL-17 induction of innate IL-12 and IL-23 production.</a> Journal of Immunology. 186 (11), 6313-6318 (2011).</li><li>Chiaranunt, P., Tometich, J. T., Ji, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> T Cell Proliferation and Colitis Are Initiated by Defined Intestinal Microbes. 201 (1), 243-250 (2018).</li><li>Feng, T., Cao, A. T., Weaver, C. T., Elson, C. O., Cong, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interleukin-12+converts+Foxp3++regulatory+T+cells+to+interferon-&#947;-producing+Foxp3++T+cells+that+inhibit+colitis.">Interleukin-12 converts Foxp3+ regulatory T cells to interferon-&#947;-producing Foxp3+ T cells that inhibit colitis.</a> Gastroenterology. 140 (7), 2031-2043 (2011).</li></ol>

No authors have conflicting financial, professional, or personal interests.

Although every step is essential for the reproducibility of this colitis model, there are several critical steps. The recipient Rag-/- mice should receive adequate viable na&#970;ve CD4+ T cells to induce intestinal inflammation. We used spleens for the isolation of na&#239;ve CD4+ T cells instead of MLNs. Because the yield of na&#239;ve CD4+ T cells in MLNs is much lower than in spleens. CD62L is highly expressed in na&#239;ve T cells, and CD44 and CD25 are the activa...

Inflammatory bowel diseases (IBD), mainly including Crohn&#39;s disease (CD) and ulcerative colitis (UC), are characterized by chronic, relapsing-remitting inflammation of the gastrointestinal tract, affecting millions worldwide1. Several factors have been implicated in the development and pathogenesis of IBD, including genetic susceptibility, gut microbiota, immune responses, diet, and lifestyle2. However, the exact mechanism of IBD is still not completely understood.
One of the particular interests is the interaction between gut microbiota and host immune responses in regulating intestinal inflammation3. Gut microbiota provides a series of immunostimulatory molecules and antigens, which can activate immune responses4. While the balance between effector T cells and regulatory T cells (Tregs) is critical in maintaining intestinal homeostasis, the excessive intestinal mucosal CD4+ T cell response to gut microbiota antigens contributes to intestinal inflammation5,6,7. As an immunodominant gut microbiota antigen, CBir1 flagellin has been related to the pathogenesis of human CD8,9. Furthermore, transfer of CBir1 TCR transgenic (Tg) T cells induces intestinal inflammation in immune-deficient mice6, closely resembling the human IBD, indicating that this T cell transfer model helps investigate the mechanisms of human IBD.
This work describes the detailed protocol of inducing colitis in Rag1-/- mice by adoptive transfer of CBir1 TCR Tg na&#970;ve CD4+ T cells and assessing disease severity. Besides, the anticipated results are shown, and the critical steps of the procedure and troubleshooting are discussed, which will help researchers investigate the mechanisms of pathogenesis of intestinal inflammation and test the potential drugs for treating IBD.

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			<td>0.22 &#181;m vacuum-driven disposable bottle top filter</td>
			<td>MilliporeSigma</td>
			<td>SCGPS05RE</td>
			<td></td>
		</tr>
		<tr>
			<td>100x Penicillin-Streptomycin</td>
			<td>Corning</td>
			<td>30-002-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>100-&#181;m strainer</td>
			<td>BD Biosciences</td>
			<td>352360</td>
			<td></td>
		</tr>
		<tr>
			<td>3-mL Transfer Pipette</td>
			<td>Fisherbrand</td>
			<td>13-711-9CM</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Mouse CD16/32</td>
			<td>Biolegend</td>
			<td>101302</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Mouse CD25-Percp/Cy5.5</td>
			<td>Biolegend</td>
			<td>102030</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse CD3-Percp/Cy5.5</td>
			<td>Biolegend</td>
			<td>100327</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Mouse CD4 APC</td>
			<td>Biolegend</td>
			<td>100516</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Mouse CD4 Magnetic Particles</td>
			<td>BD Biosciences</td>
			<td>551539</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Mouse CD4-BV421</td>
			<td>Biolegend</td>
			<td>100544</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Mouse CD62L-PE</td>
			<td>Biolegend</td>
			<td>104408</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Mouse Foxp3-PE</td>
			<td>ThermoFisher</td>
			<td>12-5773-82</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Mouse IFN&#947;-FITC</td>
			<td>Biolegend</td>
			<td>505806</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Mouse IL-17A-PE/Cy7</td>
			<td>Biolegend</td>
			<td>506922</td>
			<td></td>
		</tr>
		<tr>
			<td>Automated Cell Counter</td>
			<td>Bio-rad</td>
			<td>TC20</td>
			<td></td>
		</tr>
		<tr>
			<td>Brefeldin A</td>
			<td>BD Biosciences</td>
			<td>555029</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Fisher Bioreagents</td>
			<td>BP1600-1</td>
			<td></td>
		</tr>
		<tr>
			<td>C tube</td>
			<td>Miltenyi</td>
			<td>130-093-237</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Separation Magnet</td>
			<td>BD Biosciences</td>
			<td>552311</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase IV</td>
			<td>Sigma-Aldrich</td>
			<td>C5138</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Sigma-Aldrich</td>
			<td>D9542</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissociator Machine</td>
			<td>Miltenyi</td>
			<td>130-096-427</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase I</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Corning</td>
			<td>46-034-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA (0.5 M, PH 8.0)</td>
			<td>Corning</td>
			<td>46-034-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>R&#38;D Systems</td>
			<td>S11550</td>
			<td></td>
		</tr>
		<tr>
			<td>Flow cytometer</td>
			<td>BD Biosciences</td>
			<td>LSD Fortessa</td>
			<td></td>
		</tr>
		<tr>
			<td>Heat Lamp</td>
			<td>CoverShield</td>
			<td>BR40</td>
			<td></td>
		</tr>
		<tr>
			<td>Hematoxylin and Eosin (H&#38;E) Stain Kit</td>
			<td>Abcam</td>
			<td>ab245880</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin Syringes</td>
			<td>BD Biosciences</td>
			<td>329412</td>
			<td></td>
		</tr>
		<tr>
			<td>Ionomycin</td>
			<td>ThermoFisher</td>
			<td>I24222</td>
			<td></td>
		</tr>
		<tr>
			<td>Live/dead Fixable Near-IR Dead Cell Stain kit</td>
			<td>ThermoFisher</td>
			<td>L10119</td>
			<td></td>
		</tr>
		<tr>
			<td>MaxQ 6000 Incubated/Refrigerated Stackable Shakers</td>
			<td>ThermoFisher</td>
			<td>SHKE6000</td>
			<td></td>
		</tr>
		<tr>
			<td>NH4Cl</td>
			<td>Thermo Scientific</td>
			<td>A687-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Percoll</td>
			<td>GE Healthcare</td>
			<td>17-0891-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Phorbol-12-myristate 13-acetate</td>
			<td>Sigma-Aldrich</td>
			<td>P8139</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640 Medium</td>
			<td>Cytiva HyClone</td>
			<td>SH3002702</td>
			<td></td>
		</tr>
		<tr>
			<td>Sorter</td>
			<td>BD Biosciences</td>
			<td>Arial Fusion</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Automatic Processor</td>
			<td>ThermoFisher</td>
			<td>STP120</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Embedding/Processing Cassette</td>
			<td>Fisher Healthcare</td>
			<td>22048142</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris Base</td>
			<td>Thermo Scientific</td>
			<td>BP154-1</td>
			<td></td>
		</tr>
		<tr>
			<td>True-Nuclear Transcription Factor Buffer Set (including Perm Buffer)</td>
			<td>Biolegend</td>
			<td>424401</td>
			<td></td>
		</tr>
	</tbody>
</table>

induction of intestinal inflammation by adoptive transfer of cbir1 tcr transgenic cd4+ t cells to immunodeficient mice

With the increase of incidence, inflammatory bowel diseases (IBD), which are chronic diseases affecting the gastrointestinal tract, impose a considerable health and financial burden on individuals and society. Therefore, it is critical to investigate the mechanisms underlying the pathogenesis and development of IBD. Here, a gut microbiota antigen-specific T cell transfer colitis model is described. CBir1 flagellin has been recognized as the immunodominant gut bacterial antigen in experimental colitis and patients with Crohn&#39;s disease. CBir1 TCR transgenic na&#970;ve CD4+ T cells, specific to CBir1 flagellin, can induce chronic colitis after adoptive transfer into immune-deficient Rag1-/- mice. The disease severity is assessed by histopathology. The CD4+ T cell phenotypes in colonic lamina propria are also determined. This model closely resembles the development of IBD, which provides an ideal murine model for investigating the mechanisms driving the pathogenesis of IBD and testing the potential drugs for treating IBD.

Approximately 5 x 106 CBir1 TCR Tg na&#970;ve CD4+ T cells per spleen were isolated from an adult CBir1 TCR Tg mouse. Transfer of CBir1 TCR Tg na&#970;ve CD4+ T cells induced chronic colitis in recipient Rag1-/- mice. After cell transfer, clinical signs were monitored to evaluate the progression of intestinal inflammation, including weight loss, stool consistency, and hunched posture. As expected, mice began to lose weight around three weeks post cell transfer, and the...

Watch this Scientific Journal Video about Induction of Intestinal Inflammation by Adoptive Transfer of CBir1 TCR Transgenic CD4+ T Cells to Immunodeficient Mice at JoVE.com

Induction of Intestinal Inflammation by Adoptive Transfer of CBir1 TCR Transgenic CD4+ T Cells to Immunodeficient Mice

In this protocol, a gut microbiota antigen-specific T cell adoptive transfer colitis model is described. CD4+ T cells are isolated from CBir1 TCR transgenic mice. These are specific for an immunodominant gut microbiota antigen CBir1 flagellin, which is transferred into recipient Rag1-/- mice, leading to intestinal inflammation.

Induction of Intestinal Inflammation by Adoptive Transfer of CBir1 TCR Transgenic CD4+ T Cells to Immunodeficient Mice

With the increase of incidence, inflammatory bowel diseases (IBD), which are chronic diseases affecting the gastrointestinal tract, impose a considerable ...

induction-intestinal-inflammation-adoptive-transfer-cbir1-tcr

Research

JoVE Journal

Medicine

2.4K Views.  University of Texas Medical Branch. In this protocol, a gut microbiota antigen-specific T cell adoptive transfer colitis model is described. CD4+ T cells are isolated from CBir1 TCR transgenic mice. These are specific for an immunodominant gut microbiota antigen CBir1 flagellin, which is transferred into recipient Rag1-/- mice, leading to intestinal inflammation.

Video: Induction of Intestinal Inflammation by Adoptive Transfer of CBir1 TCR Transgenic CD4+ T Cells to Immunodeficient Mice

Functional Assessment of Intestinal Motility and Gut Wall Inflammation in Rodents: Analyses in a Standardized Model of Intestinal Manipulation

Inflammation of the gastrointestinal tract is a common reason for a variety of human diseases. Animal research models are critical in investigating the complex cellular and molecular of intestinal pathology. Although the tunica mucosa is often the organ of interest in many inflammatory diseases, recent works demonstrated that the muscularis externa (ME) is also a highly immunocompetent organ that harbours a dense network of resident immunocytes.1,2 These works were performed within the standardized model of intestinal manipulation (IM) that leads to inflammation of the bowel wall, mainly limited to the ME. Clinically this inflammation leads to prolonged intestinal dysmotility, known as postoperative ileus (POI) which is a frequent and unavoidable complication after abdominal surgery.3 The inflammation is characterized by liberation of proinflammatory mediators such as IL-64 or IL-1&beta; or inhibitory neurotransmitters like nitric oxide (NO).5 Subsequently, tremendous numbers of immunocytes extravasate into the ME, dominated by polymorphonuclear neutrophils (PMN) and monocytes and finally maintain POI.2 Lasting for days, this intestinal paralysis leads to an increased risk of aspiration, bacterial translocation and infectious complications up to sepsis and multi organ failure and causes a high economic burden.6
In this manuscript we demonstrate the standardized model of IM and in vivo assessment of gastrointestinal transit (GIT) and colonic transit. Furthermore we demonstrate a method for separation of the ME from the tunica mucosa followed by immunological analysis, which is crucial to distinguish between the inflammatory responses in these both highly immunoactive bowel wall compartments. All analyses are easily transferable to any other research models, affecting gastrointestinal function.

Postoperative ileus (POI) is a complication of abdominal surgery leading to increased morbidity and a prolonged hospital stay. Because prophylactic or therapeutic strategies are lacking intensified research is necessary. Therefore we established a standardized and feasible mouse model to investigate the pathophysiology of POI and to study potential therapeutic options.

Inflammation of the gastrointestinal tract is a common reason for a variety of human diseases. Animal research models are critical in investigating the ...

Neo-Islet Formation in Liver of Diabetic Mice by Helper-dependent Adenoviral Vector-Mediated Gene Transfer

Type 1 diabetes is caused by T cell-mediated autoimmune destruction of insulin-producing cells in the pancreas. Until now insulin replacement is still the major therapy, because islet transplantation has been limited by donor availability and by the need for long-term immunosuppression. Induced islet neogenesis by gene transfer of Neuogenin3 (Ngn3), the islet lineage-defining specific transcription factor and Betacellulin (Btc), an islet growth factor has the potential to cure type 1 diabetes.
Adenoviral vectors (Ads) are highly efficient gene transfer vector; however, early generation Ads have several disadvantages for in vivo use. Helper-dependent Ads (HDAds) are the most advanced Ads that were developed to improve the safety profile of early generation of Ads and to prolong transgene expression1. They lack chronic toxicity because they lack viral coding sequences2-5 and retain only Ad cis elements necessary for vector replication and packaging. This allows cloning of up to 36 kb genes.
In this protocol, we describe the method to generate HDAd-Ngn3 and HDAd-Btc and to deliver these vectors into STZ-induced diabetic mice. Our results show that co-injection of HDAd-Ngn3 and HDAd-Btc induces 'neo islets' in the liver and reverses hyperglycemia in diabetic mice.

We describe hepatic neo-islet formation in STZ (streptozotocin)-induced diabetic mice by gene transfer of Neurogenin3 (Ngn3) and Betacellulin (Btc) using helper-dependent adenoviral vector (HDAd) and the reversal of hyperglycemia. Our method takes advantages of helper-dependent adenoviral vectors with their highly efficient in vivo transduction and the long lasting gene expression.

Type 1 diabetes is caused by T cell-mediated autoimmune destruction of insulin-producing cells in the pancreas. Until now insulin replacement is still the ...

Generation of Subcutaneous and Intrahepatic Human Hepatocellular Carcinoma Xenografts in Immunodeficient Mice

In vivo experimental models of hepatocellular carcinoma (HCC) that recapitulate the human disease provide a valuable platform for research into disease pathophysiology and for the preclinical evaluation of novel therapies. We present a variety of methods to generate subcutaneous or orthotopic human HCC xenografts in immunodeficient mice that could be utilized in a variety of research applications. With a focus on the use of primary tumor tissue from patients undergoing surgical resection as a starting point, we describe the preparation of cell suspensions or tumor fragments for xenografting. We describe specific techniques to xenograft these tissues i) subcutaneously; or ii) intrahepatically, either by direct implantation of tumor cells or fragments into the liver, or indirectly by injection of cells into the mouse spleen. We also describe the use of partial resection of the native mouse liver at the time of xenografting as a strategy to induce a state of active liver regeneration in the recipient mouse that may facilitate the intrahepatic engraftment of primary human tumor cells. The expected results of these techniques are illustrated. The protocols described have been validated using primary human HCC samples and xenografts, which typically perform less robustly than the well-established human HCC cell lines that are widely used and frequently cited in the literature. In comparison with cell lines, we discuss factors which may contribute to the relatively low chance of primary HCC engraftment in xenotransplantation models and comment on technical issues that may influence the kinetics of xenograft growth. We also suggest methods that should be applied to ensure that xenografts obtained accurately resemble parent HCC tissues.

Human tumor xenografts in immunodeficient mice are valuable tools to study cancer biology. Specific protocols to generate subcutaneous and intrahepatic xenografts from human hepatocellular carcinoma cells or tumor fragments are described. Liver regeneration induced by partial hepatectomy in recipient mice is presented as a strategy to facilitate intrahepatic engraftment.

In  vivo experimental models of hepatocellular carcinoma (HCC) that recapitulate  the human disease provide a valuable platform for research into disease  ...

Induction of Invasive Transitional Cell Bladder Carcinoma in Immune Intact Human MUC1 Transgenic Mice:  A Model for Immunotherapy Development

A preclinical model of invasive bladder cancer was developed in human mucin 1 (MUC1) transgenic (MUC1.Tg) mice for the purpose of evaluating immunotherapy and/or cytotoxic chemotherapy. To induce bladder cancer, C57BL/6 mice (MUC1.Tg and wild type) were treated orally with the carcinogen N-butyl-N-(4-hydroxybutyl)nitrosamine (OH-BBN) at 3.0 mg/day, 5 days/week for 12 weeks. To assess the effects of OH-BBN on serum cytokine profile during tumor development, whole blood was collected via submandibular bleeds prior to treatment and every four weeks. In addition, a MUC1-targeted peptide vaccine and placebo were administered to groups of mice weekly for eight weeks. Multiplex fluorometric microbead immunoanalyses of serum cytokines during tumor development and following vaccination were performed. At termination, interferon gamma (IFN-&#947;)/interleukin-4 (IL-4) ELISpot analysis for MUC1 specific T-cell immune response and histopathological evaluations of tumor type and grade were performed. The results showed that: (1) the incidence of bladder cancer in both MUC1.Tg and wild type mice was 67%; (2) transitional cell carcinomas (TCC) developed at a 2:1 ratio compared to squamous cell carcinomas (SCC); (3) inflammatory cytokines increased with time during tumor development; and (4) administration of the peptide vaccine induces a Th1-polarized serum cytokine profile and a MUC1 specific T-cell response. All tumors in MUC1.Tg mice were positive for MUC1 expression, and half of all tumors in MUC1.Tg and wild type mice were invasive. In conclusion, using a team approach through the coordination of the efforts of pharmacologists, immunologists, pathologists and molecular biologists, we have developed an immune intact transgenic mouse model of bladder cancer that expresses hMUC1.

An N-butyl-N-(4-hydroxybutyl)nitrosamine-induced bladder cancer model was developed in human mucin 1 (MUC1) transgenic mice for the purpose of testing MUC1-directed immunotherapy. After administering a MUC1-targeted peptide vaccine, a cytotoxic T lymphocyte response to MUC1 was confirmed by measuring serum cytokine levels and T-cell specific activity.

A preclinical model of invasive bladder cancer was developed in human mucin 1 (MUC1) transgenic (MUC1.Tg) mice for the purpose of evaluating immunotherapy ...

Murine Endoscopy for In Vivo Multimodal Imaging of Carcinogenesis and Assessment of Intestinal Wound Healing and Inflammation

Mouse models are widely used to study pathogenesis of human diseases and to evaluate diagnostic procedures as well as therapeutic interventions preclinically. However, valid assessment of pathological alterations often requires histological analysis, and when performed ex vivo, necessitates death of the animal. Therefore in conventional experimental settings, intra-individual follow-up examinations are rarely possible. Thus, development of murine endoscopy in live mice enables investigators for the first time to both directly visualize the gastrointestinal mucosa and also repeat the procedure to monitor for alterations. Numerous applications for in vivo murine endoscopy exist, including studying intestinal inflammation or wound healing, obtaining mucosal biopsies repeatedly, and to locally administer diagnostic or therapeutic agents using miniature injection catheters. Most recently, molecular imaging has extended diagnostic imaging modalities allowing specific detection of distinct target molecules using specific photoprobes. In conclusion, murine endoscopy has emerged as a novel cutting-edge technology for diagnostic experimental in vivo imaging and may significantly impact on preclinical research in various fields.

Murine Endoscopy for In Vivo Multimodal Imaging of Carcinogenesis and Assessment of Intestinal Wound Healing and Inflammation

Small animal imaging techniques allow serial diagnostic examinations and therapeutic interventions in vivo. Recently, the scope of applications has significantly widened and currently includes assessment of colonic tumor development, wound healing and monitoring of inflammation. This protocol illustrates these diverse potential applications of murine endoscopy.

Mouse models are widely used to study pathogenesis of human diseases and to evaluate diagnostic procedures as well as therapeutic interventions ...

Bile Duct Ligation in Mice: Induction of Inflammatory Liver Injury and Fibrosis by Obstructive Cholestasis

In most vertebrates, the liver produces bile that is necessary to emulsify absorbed fats and enable the digestion of lipids in the small intestine as well as to excrete bilirubin and other metabolic products. In the liver, the experimental obstruction of the extrahepatic biliary system initiates a complex cascade of pathological events that leads to cholestasis and inflammation resulting in a strong fibrotic reaction originating from the periportal fields. Therefore, surgical ligation of the common bile duct has become the most commonly used model to induce obstructive cholestatic injury in rodents and to study the molecular and cellular events that underlie these pathophysiological mechanisms induced by inappropriate bile flow. In recent years, different surgical techniques have been described that either allow reconnection or reanastomosis after bile duct ligation (BDL), e.g., partial BDL, or other microsurgical methods for specific research questions. However, the most frequently used model is the complete obstruction of the common bile duct that induces a strong fibrotic response after 21 to 28 days. The mortality rate can be high due to infectious complications or technical inaccuracies. Here we provide a detailed surgical procedure for the BDL model in mice that induce a highly reproducible fibrotic response in accordance to the 3R rule for animal welfare postulated by Russel and Burch in 1959.

Disruption of bile flow results in severe inflammatory cholestatic liver injury with a characteristic time-dependent sequence of morphological alterations. Here we present a protocol for the surgical ligation of the common bile duct in mice that allows to induce a strong fibrotic response after 21 to 28 days.

In most vertebrates, the liver produces bile that is necessary to emulsify absorbed fats and enable the digestion of lipids in the small intestine as well ...

Th17 Inflammation Model of Oropharyngeal Candidiasis in Immunodeficient Mice

Oropharyngeal Candidiasis (OPC) disease is caused not only due to the lack of host immune resistance, but also the absence of appropriate regulation of infection-induced immunopathology. Although Th17 cells are implicated in antifungal defense, their role in immunopathology is unclear. This study presents a method for establishing oral Th17 immunopathology associated with oral candidal infection in immunodeficient mice. The method is based on reconstituting lymphopenic mice with in vitro cultured Th17 cells, followed by oral infection with Candida albicans (C. albicans). Results show that unrestrained Th17 cells result in inflammation and pathology, and is associated with several measurable read-outs including weight loss, pro-inflammatory cytokine production, tongue histopathology and mortality, showing that this model may be valuable in studying OPC immunopathology. Adoptive transfer of regulatory cells (Tregs) controls and reduces the inflammatory response, showing that this model can be used to test new strategies to counteract oral inflammation. This model may also be applicable in studying oral Th17 immunopathology in general in the context of other oral diseases.

Although Candida infection models are available to study host immune resistance, a model to study T cell mediated immunopathology in the context of Candida infection is absent. Here we describe a method to establish Th17 immunopathology associated with oral Candida infection in immunodeficient mice.

Oropharyngeal Candidiasis (OPC) disease is caused not only due to the lack of host immune resistance, but also the absence of appropriate regulation of ...

Dry Powder and Nebulized Aerosol Inhalation of Pharmaceuticals Delivered to Mice Using a Nose-only Exposure System

Obstructive respiratory diseases like asthma and chronic obstructive pulmonary disease (COPD) are currently treated by inhaled anti-inflammatory and bronchodilator drugs. Despite the availability of multiple treatments, both diseases are growing public health concerns. The majority of asthma patients are well controlled on current inhaled therapies but a substantial number of patients with severe asthma are not. Asthma affects an estimated 300 million people worldwide and approximately 20 percent have a severe form of the disease. In contrast to asthma, there are few effective therapies for COPD. An estimated 10% of the population has COPD and the trend in death rates is increasing for COPD while decreasing for other major diseases. Although developing drugs for inhaled delivery is challenging, the nose-only inhalation unit enables direct delivery of novel drugs to the lung of rodents for pre-clinical efficacy and safety/toxicology studies. Inhaled drug delivery has multiple advantages for respiratory diseases, where high concentration in the lung improves efficacy and minimizes systemic side effects. Inhaled corticosteroids and bronchodilators benefit from these advantages and inhaled delivery may also hold potential for future biologic therapies. The inhalation unit described herein can generate, sample for characterization, and uniformly deposit a drug aerosol in the lungs of rodents. This enables the pre-clinical determination of the efficacy and safety of drug doses deposited in the lungs of rodents, key data required before initiating clinical development.

The inhalation unit described herein can generate, sample for characterization, and uniformly deposit a drug aerosol in the lungs of rodents. This enables the pre-clinical determination of the efficacy and safety of drug doses deposited in the lungs; key data enabling clinical inhaled drug development.

Obstructive respiratory diseases like asthma and chronic obstructive pulmonary disease (COPD) are currently treated by inhaled anti-inflammatory and ...

Fluorescence-mediated Tomography for the Detection and Quantification of Macrophage-related Murine Intestinal Inflammation

Murine models of disease are indispensable to scientific research. However, many diagnostic tools such as endoscopy or tomographic imaging are not routinely employed in animal models. Conventional experimental readouts often rely on post mortem and ex vivo analyses, which prevent intra-individual follow-up examinations and increase the number of study animals needed. Fluorescence-mediated tomography enables the non-invasive, repetitive, quantitative, three-dimensional assessment of fluorescent probes. It is highly sensitive and permits the use of molecular makers, which allows for the specific detection and characterization of distinct molecular targets. In particular, targeted probes represent an innovative tool for analyzing gene activation and protein expression in inflammation, autoimmune disease, infection, vascular disease, cell migration, tumorigenesis, etc. In this article, we provide step-by-step instructions on this sophisticated imaging technology for the in vivo detection and characterization of inflammation (i.e., F4/80-positive macrophage infiltration) in a widely used murine model of intestinal inflammation. This technique might also be used in other research areas, such as immune cell or stem cell tracking.

Target-specific probes represent an innovative tool for analyzing molecular mechanisms, such as protein expression in various types of disease (e.g., inflammation, infection, and tumorigenesis). In this study, we describe a quantitative three-dimensional tomographic assessment of intestinal macrophage infiltration in a murine model of colitis using F4/80-specific fluorescence-mediated tomography.

Murine models of disease are indispensable to scientific research. However, many diagnostic tools such as endoscopy or tomographic imaging are not ...

Induction of Nephrotic Syndrome in Mice by Retrobulbar Injection of Doxorubicin and Prevention of Volume Retention by Sustained Release Aprotinin

Nephrotic syndrome is the most extreme manifestation of proteinuric kidney disease and characterized by heavy proteinuria, hypoalbuminemia, and edema due to sodium retention and hyperlipidemia. To study the pathophysiology of this syndrome, rodent models have been developed based on the injection of toxic substances such as doxorubicin causing podocyte damage. In mice, only few strains are susceptible to this model. In wildtype 129S1/SvImJ mice, the administration of doxorubicin by rapid intravenous injection to the retrobulbar sinus induces experimental nephrotic syndrome that features all the symptoms of human disease including sodium retention and edema. After the onset of proteinuria, mice exhibit increased urinary serine protease activity that leads to the activation of the epithelial sodium channel (ENaC) and sodium retention. Pharmacological inhibition of urinary serine proteases by the treatment with sustained release aprotinin abrogates ENaC activation and prevents sodium retention. This model is ideal to study the pathophysiology of proteasuria, i.e., the excretion of active serine proteases that cause ENaC activation by the proteolysis of its &#947;-subunit. This can be regarded as the primary mechanism of ENaC activation and sodium retention in proteinuric kidney disease.

Here, we describe the induction of experimental nephrotic syndrome in 129S1/SvImJ mice by rapid retrobulbar injection of doxorubicin. We also treat nephrotic mice with sustained release pellets containing aprotinin to inhibit urinary serine protease activity and prevent sodium retention.

Nephrotic syndrome is the most extreme manifestation of proteinuric kidney disease and characterized by heavy proteinuria, hypoalbuminemia, and edema due ...