This work was supported, in part, by grants from the National Institutes of Health (R01 HL152683 and R21 AI126097 to Q. Li) and by American Heart Association Grant-in-Aid 17GRNT33460395 (to Q. Li) (heart.org).

All procedures performed on animals were approved by the Institutional Animal Care and Use Committee of the University of Texas Medical Branch at Galveston (Protocol # 1512071A).
1. Collection of fresh fecal pellets
<ol>
	<li>Prepare sterile paper towels, blunt-end forceps, and 50 mL conical tubes.
	<ol>
		<li>Place some paper towels and forceps in separate autoclave bags and autoclave them at 180 &#176;C in dry heat for 30 min. Use sterile conical tubes as well. Weigh the conical tubes and write down their weight on the tubes.</li>
	</ol>
	</li>
	<li>Turn on the biosafety cabinet in the animal room.</li>
	<li>Take an autoclaved clean mouse cage without any bedding and place it in the biosafety cabinet. Remove the cover and the food rack and place them inside the cabinet.</li>
	<li>Place some sterile paper towels on the bottom of the cage and place the metal rack back on top of the cage.</li>
	<li>Identify age-matched fecal donors and place the mouse cage in the biosafety cabinet. Open the cage and gently grab a donor mouse (C57BL/6J) by the tail and place it on the metal rack on top of the clean cage.</li>
	<li>Place the cage cover on top of the rack and wait for the animal(s) to defecate. 
	NOTE: Put a few mouse littermates on the rack simultaneously.</li>
	<li>Collect the fecal pellets and put them in a sterile 50 mL conical tube. Pool the pellets by sex. Do not mix fecal pellets collected from males and females.</li>
	<li>Weight the tube again and calculate the weight of the fecal pellets.</li>
</ol>
2. Preparation of fecal suspension
<ol>
	<li>Prepare a sterile solution (10% glycerol in normal saline).</li>
	<li>Add 10 mL of 10% glycerol/normal saline to the conical tube for each gram of fecal pellets. 
	NOTE: Increase the solution volume to 20 mL if necessary. This study used 1 mL of solution for each pellet (5-10 mg) as well.</li>
	<li>Homogenize the mixture at a low speed with a benchtop homogenizer or a blender inside a fume hood to resuspend the feces (3 X 30 s).</li>
	<li>Filter the fecal suspension through 2 layers of sterile cotton gauze (10.2 cm x 10.2 cm). Store the filtrate temporarily in a refrigerator for up to 6 h or package it into sterile cryogenic vials and store it in a -80 &#176;C freezer.</li>
	<li>Thoroughly clean the homogenizer or blender following a standard procedure.</li>
</ol>
3. Administration of fecal suspension by oral gavage
<ol>
	<li>Thaw the frozen fecal suspension on ice if using frozen samples. Mix the thawed fecal suspension by vortexing.</li>
	<li>Transfer the fresh or thawed fecal suspension to 1 mL syringes. 
	NOTE: Each mouse will receive a total of 200 &#181;L of fecal suspension, and each IL-10-/- mouse in the control group will get 200 &#181;L of 10% glycerol/normal saline17.</li>
	<li>Weigh the mice and choose the right gavage needle size and maximum dosage volume. 
	NOTE: For mice with a bodyweight between 20-25 grams, use a 20 G 3.81 cm curved gavage needle with a 2.25 mm ball. Please check gavageneedle.com for more information.</li>
	<li>Test the gavage needle by measuring the length from the tip of the mouse&#39;s nose to the xiphoid process (bottom of the sternum). Fill the syringe with 10% glycerol/saline or fecal suspension and remove air bubbles inside the syringe and the needle. 
	NOTE: If the needle is longer than the length, put a mark on the needle shaft/tubing at the level of the nose. Do not pass the needle/tubing through the animal past that point to prevent gastric perforation.</li>
	<li>Place one mouse cage in the biosafety cabinet, remove the plastic cage cover, and leave the metal rack in place.</li>
	<li>Grab one mouse by the tail and put it on the metal rack. Hold the mouse by the tail with one hand and use the thumb and middle fingers of another hand to restrain the animal by grasping the skin over the shoulders. This way, the forelegs are stretched out to the side, which will prevent the front feet from pushing the needle out. Gently extend the animal&#39;s head backward and hold the head in place with one hand. 
	NOTE: Practice mouse handling until the experimenter has full confidence before proceeding to the experiment.</li>
	<li>Place the gavage needle on top of the tongue inside the mouth. Gently advance along the upper palate until the needle reaches the esophagus. Pass the needle smoothly in one motion. Do not force the needle if any resistance is felt. Take the needle out and try again.</li>
	<li>Once the needle is properly placed and verified, slowly administer the material by pushing the syringe attached to the needle. Do not rotate the needle or push the needle forward, which may rupture the esophagus. After dosing, gently pull the needle out.</li>
	<li>Return the mouse to its home cage. Monitor the animal for 5-10 min by looking for signs of labored breathing or distress. Monitor the mice again between 12-24 h after the FMT.</li>
</ol>
4. Disease monitoring and euthanasia
<ol>
	<li>Monitor the mice longitudinally for IBD onset by occult fecal blood and/or colonoscopy21. Evaluate heart function by trans-thoracic echocardiography22,23.</li>
	<li>Euthanize the animals by decapitation under a deep plane of anesthesia with isoflurane (1%-4%).</li>
	<li>Collect the blood in microcentrifuge tubes with anticoagulant and centrifuge at 1000-2000 x g for 10 min in a refrigerated centrifuge (4 &#176;C). Save the supernatant, designated plasma in a -80&#176;C freezer.</li>
	<li>Upon euthanization, prepare the mouse colon using the Swiss-roll technique24 for histopathologic analysis (H&#38;E staining)25.</li>
	<li>Measure B-type natriuretic peptide (BNP) in plasma using an enzyme immunoassay (EIA) kit23.</li>
</ol>

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FDA Available from: <a target="_blank" href="https://www.fda.gov/vaccines-blood-biologics/safety-availability-biologics/safety-alert-regarding-use-fecal-microbiota-tramsplantation-and-risk-serious-adverse-events-likely">https://www.fda.gov/vaccines-blood-biologics/safety-availability-biologics/safety-alert-regarding-use-fecal-microbiota-tramsplantation-and-risk-serious-adverse-events-likely</a> (2020)</li></ol>

The authors declare that they have no competing interests.

As an innovative investigational treatment, FMT has become a hot topic in the treatment of various disorders in recent years since dysbiosis of the commensal microbiota is implicated in the pathogenesis of multiple human diseases, including IBD, obesity, diabetes mellitus, autism, heart disease, and cancer26. Although the mechanism has not been determined, it is believed that FMT works by building a new biological flora and preventing the loss of residual bacteria. The method presented herein adop...

The human intestinal micro-ecosystem is extremely complex, with more than 1000 species of bacteria in the intestine of a healthy person1. The intestinal flora is involved in maintaining the normal physiological functions of the intestine and the immune response and has an inseparable relationship with the human body. Accumulating evidence suggests that the intestinal microbiome constitutes the last human organ, which is part of the human body, not just a group of parasites2. A &#39;healthy&#39; symbiotic relationship between the gut microbiota, their metabolites, and the host immune system established in early life is critical to maintaining gut homeostasis. In some abnormal conditions such as chronic inflammation, changes in the internal and external environment of the body seriously disrupt the gut homeostasis, resulting in a persistent imbalance of the gut&#39;s microbial community, named dysbiosis3. In fact, exposure to multiple environmental factors, including diet, drugs, and pathogens, can lead to changes in the microbiota.
Dysbiosis is associated with the pathogenesis of a variety of intestinal diseases, such as inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), and pseudomembranous enteritis, as well as a growing list of extra-intestinal disorders, including cardiovascular disease, obesity, and allergy4. Microbiota profiling revealed that patients with IBD have a dramatic decrease in bacterial diversity, as well as marked alterations in the populations of some specific bacterial strains5,6. These studies demonstrated less Lachnospiraceae and Bacteroidetes but more Proteobacteria and Actinobacteria in IBD patients. It is believed that the pathogenesis of IBD is related to various pathogenic factors, including abnormal intestinal flora, dysregulated immune response, environmental challenges, and genetic variants7.Abundant evidence suggests that intestinal bacteria play a role in the initiation and application phases of IBD8,9, indicating that correcting gut dysbiosis may represent a novel approach for the therapy and/or maintenance treatment of IBD.
The prototype of fecal microbiota transplantation (FMT) began in ancient China10. In 1958, Dr. Eiseman and his colleagues successfully treated four cases of severe pseudomembranous enteritis with fecal matter from healthy donors via enema, opening a new chapter in modern Western medicine using human feces to treat human diseases11. Clostridium difficile infection (CDI) has been found to be the main cause of pseudomembranous enteritis12 and FMT is highly effective in the treatment of CDI. In the last eight years, FMT has become a standard-of-care therapy for the treatment of recurrent CDI13, prompting further studies investigating the role of FMT in other disorders, such as IBD. Over the past twenty years, numerous case reports and cohort studies have documented the use of FMT in patients with IBD14. A meta-analysis, including 12 trials, showed that 62% of patients with Crohn&#39;s disease (CD) achieved clinical remission after FMT, and 69% of CD patients had clinical response15. Despite these encouraging findings, the role of FMT in the management of IBD remains uncertain, and the mechanisms by which FMT ameliorates intestinal inflammation are poorly understood. Further investigation is necessary before FMT can join the current armamentarium of treatment options for IBD in the clinics.
In this protocol, we applied FMT on IL-10-/- mice, which develop colitis spontaneously after weaning and have served as a gold standard to mirror the multifactorial nature of IBD16,17,18.&#160;IL-10&#8722;/&#8722; mice have been extensively used to dissect IBD etiology because they present similar molecular and histological features to IBD patients, and, like patients, the disease can be ameliorated with anti-TNF&#945; therapy16. Aging IL10&#8722;/&#8722; mice (&#62;9 months of age) have an increased heart size and impaired cardiac function compared to age-matched wild-type mice19, making it an excellent model for studying colitis-induced heart diseases. However, other murine models of colitis, such as the dextran sodium sulfate model and the T-cell-induced colitis model, can be used as well. We administered fecal suspension via oral gavage, proven to be an effective and better route than enema in humans20.

<table><tbody><tr><td>BD Syringe, 1 mL</td><td>Fisher Scientific</td><td>14-829-10F</td><td></td></tr><tr><td>Blunt end forceps</td><td>Knipex</td><td>926443</td><td></td></tr><tr><td>Brain natriuretic peptide EIA kit</td><td>Sigma</td><td>RAB0386</td><td></td></tr><tr><td>C57BL/6J mice</td><td>Jackson Lab</td><td>000664</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5415R</td><td></td></tr><tr><td>Conical tubes</td><td>ThermoFisher</td><td>339650</td><td></td></tr><tr><td>Curved feeding Needles</td><td>Kent Scientific</td><td>FNC-20-1.5-2</td><td></td></tr><tr><td>GLH-115 homogenizer</td><td>Omni International</td><td>GLH-115</td><td></td></tr><tr><td>Glycerol</td><td>MilliporeSigma</td><td>G5516</td><td></td></tr><tr><td>IL-10 knockout mice</td><td>Jackson Lab</td><td>004366</td><td></td></tr><tr><td>Isoflurane</td><td>Piramal Critical care</td><td>NDC66794-017-10</td><td></td></tr><tr><td>USP normal saline</td><td>Grainger</td><td>6280</td><td></td></tr><tr><td>Vaporizer</td><td>Euthanex Corp.</td><td>EZ-108SA</td><td></td></tr></tbody></table>

therapeutic evaluation of fecal microbiota transplantation in an interleukin 10-deficient mouse model

With the development of microecology in recent years, the relationship between intestinal bacteria and inflammatory bowel disease (IBD) has attracted considerable attention. Accumulating evidence suggests that dysbiotic microbiota plays an active role in triggering or worsening the inflammatory process in IBD and that fecal microbiota transplantation (FMT) is an attractive therapeutic strategy since transferring a healthy microbiota to IBD patient could restore the appropriate host-microbiota communication. However, the molecular mechanisms are unclear, and the efficacy of FMT has not been very well established. Thus, further studies in animal models of IBD are necessary. In this method, we applied FMT from wild-type C57BL/6J mice to IL-10 deficient mice, a widely used mouse model of colitis. The study elaborates on collecting fecal pellets from the donor mice, making the fecal solution/suspension, administering the fecal solution, and monitoring the disease. We found that FMT significantly mitigated the cardiac impairment in IL-10 knockout mice, underlining its therapeutic potential for IBD management.

We performed healthy donor FMT 3 times (once a month for 3 months) on 2-month old C57BL/6J wild type (WT) and IL-10 knockout mice. Age-matched C57BL/6J mice (age difference should be &#60;2 months) served as the fecal donors and fresh fecal pellets were used each time. EIA assays revealed that BNP was markedly elevated in the plasma of IL-10-deficiency mice and that healthy donor FMT significantly mitigated the increase in BNP levels (Figure 1A, n = 5, p &#60; 0.05). Echocardiograph...

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Therapeutic Evaluation of Fecal Microbiota Transplantation in an Interleukin 10-Deficient Mouse Model

The interaction of genetic susceptibility, mucosal immunity, and intestinal microecological environment is involved in the pathogenesis of inflammatory bowel disease (IBD). In this study, we applied fecal microbiota transplantation to IL-10 deficient mice and investigated its impact on colonic inflammation and heart function.

With the development of microecology in recent years, the relationship between intestinal bacteria and inflammatory bowel disease (IBD) has attracted ...

therapeutic-evaluation-fecal-microbiota-transplantation-an

Research

JoVE Journal

Immunology and Infection

2.6K Views.  University of Texas Medical Branch at Galveston. Drie klinische experimenten suggereerden dat fecale microbiota-transplantatie het therapeutische potentieel heeft voor een aantal menselijke ziekten. Verdere mechanistische studies zullen helpen om FMT van bank naar bed te brengen. Deze methode neemt orale maagsonde aan voor de leveringsroute, waardoor een nauwkeurigere volumelevering en de snelle absorptie mogelijk is.Het vereist slechts matige technische vaardigheden. De procedure wordt gedemonstreerd door Xioying Zhong, een senior o...

Video: Therapeutic Evaluation of Fecal Microbiota Transplantation in an Interleukin 10-Deficient Mouse Model

A Double Humanized BLT-mice Model Featuring a Stable Human-Like Gut Microbiome and Human Immune System

Humanized mice (hu-mice) that feature a functional human immune system have fundamentally changed the study of human pathogens and disease. They can be used to model diseases that are otherwise difficult or impossible to study in humans or other animal models. The gut microbiome can have a profound impact on human health and disease. However, the murine gut microbiome is very different than the one found in humans. &#160;There is a need for improved pre-clinical hu-mice models that have an engrafted human gut microbiome. Therefore, we created double hu-mice that feature both a human immune system and stable human-like gut microbiome. NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice are one of the best animals for humanization due to their high level of immunodeficiency. However, germ-free NSG mice, and various other important germ-free mice models are not currently commercially available. Further, many research settings do not have access to gnotobiotic facilities, and working under gnotobiotic conditions can often be expensive and time consuming. Importantly, germ-free mice have several immune deficiencies that exist even after the engraftment of microbes. Therefore, we developed a protocol that does not require germ-free animals or gnotobiotic facilities. To generate double hu-mice, NSG mice were treated with radiation prior to surgery to create bone-marrow, liver, thymus-humanized (hu-BLT) mice. The mice were then treated with broad spectrum antibiotics to deplete the pre-existing murine gut microbiome. After antibiotic treatment, the mice were given fecal transplants with healthy human donor samples via oral gavage. Double hu-BLT mice had unique 16S rRNA gene profiles based on the individual human donor sample that was transplanted. Importantly, the transplanted human-like microbiome was stable in the double hu-BLT mice for the duration of the study up to 14.5 weeks post-transplant.

We describe a novel method for generating double humanized BLT-mice that feature a functional human immune system and a stable engrafted human-like gut microbiome. This protocol can be followed without the need for germ-free mice or gnotobiotic facilities.

Humanized mice (hu-mice) that feature a functional human immune system have fundamentally changed the study of human pathogens and disease. They can be ...

Biochemistry

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 and disadvantages. We describe here an experimental colitis model that is initiated by adoptive transfer of syngeneic splenic CD4+CD45RBhigh T cells into T and B cell deficient recipient mice. The CD4+CD45RBhigh T cell population that largely consists of na&#239;ve effector cells is capable of inducing chronic intestinal inflammation, closely resembling key aspects of human IBD. This method can be manipulated to study aspects of disease onset and progression. Additionally it can be used to study the function of innate, adaptive, and regulatory immune cell populations, and the role of environmental exposures, i.e., the microbiota, in intestinal inflammation. In this article we illustrate the methodology for inducing colitis with a step-by-step protocol. This includes a video demonstration of key technical aspects required to successfully develop this murine model of experimental colitis for research purposes.

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

Here, we present a protocol to induce colonic inflammation in mice by adoptive transfer of syngeneic CD4+CD45RBhigh T cells into T and B cell deficient recipients. Clinical and histopathological features mimic human inflammatory bowel diseases. This method allows the study of the initiation of colonic inflammation and progression of disease.

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

Fecal Microbiota Transplantation via Colonoscopy for Recurrent C. difficile Infection

Fecal Microbiota Transplantation (FMT) is a safe and highly effective treatment for recurrent and refractory C. difficile infection (CDI). Various methods of FMT administration have been reported in the literature including nasogastric tube, upper endoscopy, enema and colonoscopy. FMT via colonoscopy yields excellent cure rates and is also well tolerated. We have found that patients find this an acceptable and tolerable mode of delivery. At our Center, we have initiated a fecal transplant program for patients with recurrent or refractory CDI. We have developed a protocol using an iterative process of revision and have performed 24 fecal transplants on 22 patients with success rates comparable to the current published literature. A systematic approach to patient and donor screening, preparation of stool, and delivery of the stool maximizes therapeutic success. Here we detail each step of the FMT protocol that can be carried out at any endoscopy center with a high degree of safety and success.

Fecal Microbiota Transplantation via Colonoscopy for Recurrent C. difficile Infection

Fecal Microbiota Transplantation via colonoscopy is a safe and effective treatment for recurrent and refractory C.difficile infection. A systematic approach to patient and donor screening, preparation of stool, and delivery of the stool during the colonoscopy will maximize therapeutic success.

Fecal Microbiota Transplantation (FMT) is a safe and highly effective treatment for recurrent and refractory C. difficile infection (CDI). Various methods ...

Medicine

Non-invasive Assessment of the Efficacy of New Therapeutics for Intestinal Pathologies Using Serial Endoscopic Imaging of Live Mice

Animal models of inflammatory bowel disease (IBD) and colorectal cancer (CRC) have provided significant insight into the cell intrinsic and extrinsic mechanisms that contribute to the onset and progression of intestinal diseases. The identification of new molecules that promote these pathologies has led to a flurry of activity focused on the development of potential new therapies to inhibit their function. As a result, various pre-clinical mouse models with an intact immune system and stromal microenvironment are now heavily used. Here we describe three experimental protocols to test the efficacy of new therapeutics in pre-clinical models of (1) acute mucosal damage, (2) chronic colitis and/or colitis-associated colon cancer, and (3) sporadic colorectal cancer. We also outline procedures for serial endoscopic examination that can be used to document the therapeutic response of an individual tumor and to monitor the health of individual mice. These protocols provide complementary experimental platforms to test the effectiveness of therapeutic compounds shown to be well tolerated by mice.

We describe methods for longitudinal monitoring of the efficacy of therapeutics for the treatment of colonic pathologies in mice using a rigid endoscope. This protocol can be readily used for the characterization of the therapeutic response of an individual tumor in live mice and also for monitoring potential disease relapse.

Animal models of inflammatory bowel disease (IBD) and colorectal cancer (CRC) have provided significant insight into the cell intrinsic and extrinsic ...

Murine Fecal Isolation and Microbiota Transplantation

Gut microbiota dysbiosis plays a role in the pathophysiology of cardiovascular and metabolic disorders, but the mechanisms are not well understood. Fecal microbiota transplantation (FMT) is a valuable approach to delineating a direct role of the total microbiota or isolated species in disease pathophysiology. It is a safe treatment option for patients with recurrent Clostridium difficile infection. Preclinical studies demonstrate that manipulating the gut microbiota is a useful tool to study the mechanistic link between dysbiosis and disease. Fecal microbiota transplantation may help elucidate novel gut microbiota-targeted therapeutics for the management and treatment of cardiometabolic disease. Despite a high success rate in rodents, there remains translational changes associated with the transplantation. The goal here is to provide guidance in studying the effects of gut microbiome in experimental cardiovascular disease. In this study, a detailed protocol for the collection, handling, processing, and transplantation of fecal microbiota in murine studies is described. The collection and processing steps are described for both human and rodent donors. Lastly, we describe using a combination of the Swiss-rolling and immunostaining techniques to assess gut-specific morphology and integrity changes in cardiovascular disease and related gut microbiota mechanisms.

The goal here is to outline a protocol to investigate the mechanisms of dysbiosis in cardiovascular disease. This paper discusses how to aseptically collect and transplant murine fecal samples, isolate intestines, and use the "Swiss-roll" method, followed by immunostaining techniques to interrogate changes in the gastrointestinal tract.

Gut microbiota dysbiosis plays a role in the pathophysiology of cardiovascular and metabolic disorders, but the mechanisms are not well understood. Fecal ...

Preparation and Maintenance of Bioexclusion IsoPositive Cage Experiment for Human Fecal Transplantation into Germ-Free Mice

Germ-free mice are an important investigation tool for understanding the contribution of microorganisms in host health and disease, enabling assessment of the specific role of individuals, defined or complex groups of microorganisms in host response. Traditionally bred and reared in flexible-film or semi-rigid isolators, germ-free mouse husbandry and experimental manipulation are costly and require numerous trained staff and a large space footprint in animal housing facilities. The IsoPositive caging system allows for experimental manipulation of germ-free mice in individual, hermetically-sealed, positive-pressure isolator cages (isocages), reducing cost and enabling greater flexibility in experimental manipulations. 
Here, a protocol is described for transferring germ-free mice from breeding isolators to isocages and subsequent fecal transfer from human donor stool into mice to create stable long-term gut "humanized" mice for future studies. The materials and preparation needed for the utilization of the isocage system are described, including the use of chlorine-dioxide sterilant chemical sterilant to clean cages, supplies, equipment, and personal protective equipment. The methods for confirming the germ-free status of transferred mice and how to determine contamination in the caging system are discussed. The procedure for husbandry, including bedding, food, and water supply, is further discussed. The protocol for human fecal slurry preparation and gavage into germ-free mice to create gut "humanized" mice, along with stool collection to monitor the microbial community composition of these mice, are described. An experiment illustrates that two weeks post-human fecal transplant allows for stable colonization of donor microbiota in the murine hosts, enabling subsequent experimental usage. Furthermore, the collection of humanized mouse feces in viability preservation media, enabling use in further functional experiments, is described. Overall, these methods allow for the safe and effective establishment of humanized mouse communities in experimental gnotobiotic cages for further manipulation.

This protocol describes the best practices for germ-free mouse transfer to and housing in experimental single-cage isolators (isocages) while maintaining sterile conditions. Methods for fecal transplant into germ-free mice and the collection of viable bacteria from these gut "humanized" mice for further applications are discussed.

Germ-free mice are an important investigation tool for understanding the contribution of microorganisms in host health and disease, enabling assessment of ...

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.

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.

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

Fecal Microbiota Transplantation and Detection of Prevalence of IgA-Coated Bacteria in the Gut

Gut microbiota exert pleiotropic roles in human health and disease. Fecal microbiota transplantation (FMT) is an effective method to investigate the biological function of intestinal bacteria as a whole or at the species level. Several different FMT methods have been published. Here, we present an FMT protocol that successfully depletes gut microbiota in a matter of days, followed by transplantation of fecal microbiota from fresh or frozen donor intestinal contents to conventional mice. Real time-PCR is applied to test the efficacy of bacterial depletion. Sequencing of the 16S ribosomal RNA (rRNA) is then applied to test the relative abundance and identity of gut microbiota in recipient mice. We also present a flow cytometry-based detection method of immunoglobulin A (IgA)-coated bacteria in the gut.

We established an efficient way to deplete intestinal bacteria in three days, and subsequently transplant fecal microbiota via gavage of fecal fluid prepared from fresh or frozen intestinal contents in mice. We also present an optimized method to detect the frequency of IgA-coated bacteria in the gut.

Gut microbiota exert pleiotropic roles in human health and disease. Fecal microbiota transplantation (FMT) is an effective method to investigate the ...

Biology

Generating a Double Humanized BLT (Bone-Marrow, Liver, Thymus) Mouse Model

Source: Daharsh, L. et al., <a href="https://app.jove.com/v/59773">A Double Humanized BLT-mice Model Featuring a Stable Human-Like Gut Microbiome and Human Immune System.</a>&#160;J. Vis. Exp. (2019)
This video demonstrates the method for generating a double humanized bone BLT (marrow, liver, and thymus) mouse with a functional human immune system and a stable engrafted human-like gut microbiome.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Mice ...

JoVE Encyclopedia of Experiments

Immunology

Immunotherapy

Model Development and Disease Study

Evaluating the Effects of Fecal Microbiota Transplantation in an Interleukin-10 Deficient Mouse

Source: Xiao, Y. et al., <a href="https://app.jove.com/v/63350">Therapeutic Evaluation of Fecal Microbiota Transplantation in an Interleukin 10-Deficient Mouse Model.</a>&#160;J. Vis. Exp. (2022)
This video demonstrates a procedure of fecal microbiota transplantation in an interleukin-10 deficient mouse with colon inflammation. Colonized fecal microbes in the gut release secondary metabolites, eliciting anti-inflammatory cytokine release and reducing colon inflammation.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

Therapeutic Evaluation of Fecal Microbiota Transplantation in an Interleukin 10-Deficient Mouse Model

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