The authors would like to acknowledge the Infectious Disease Department at Brigham and Women&#8217;s Hospital especially Michael S. Calderwood, MD, MPH for helping us construct this protocol. We would also like to thank the Institution&#8217;s endoscopy center and it&#8217;s leadership team for their support.

NOTE: At the time of this manuscript publication an Investigational New Drug (IND) application is not required to perform an FMT for recurrent CDI in clinical practice. However, if FMTs are being performed as part of a study or for another indication, an IND or equivalent application may be required18 . Regulatory agencies will likely continue to assess the safety and efficacy of FMT as applied to CDI and other medical conditions and so it is advised to check the regulatory requirements in your respective location prior to the initiation of an FMT program and every one to two months once actively performing FMT.
NOTE Candidates must be willing to consent to the fecal transplant as well as the colonoscopy.
1. Identification of Appropriate Candidates for FMT
<ol>
	<li>Patients who have relapsing CDI or patients with refractory disease that is unresponsive to traditional antibiotics are appropriate candidates for FMT. 
	NOTE: relapsing CDI is defined as a history of three or more episodes of CDI confirmed by toxin testing or PCR, or two episodes that required hospitalization.</li>
	<li>Ensure the patient is able to undergo a colonoscopy safely. This is at the discretion of the endoscopist.</li>
</ol>
2. Stool Donor Selection
NOTE: Stool Donor selection applies to the treatment of CDI and may not be applicable to the treatment of other disorders.
<ol>
	<li>Ensure the patient identifies a donor. Potential donors should be present with the recipient at the time of the initial gastroenterology clinic visit. Donors may be spouses, family members, or friends. Donors may live in the same residence as the recipient as long as other criteria are met.</li>
</ol>
3. Donor Eligibility
<ol>
	<li>Inclusion Criteria: Ensure that the donor is a healthy adult (&#8805;18 years old) and has a daily formed bowel movement.</li>
	<li>Exclusion Criteria:
	<ol>
		<li>Ensure that the donor is not taking antibiotics or has not taken antibiotics in the prior three months or any medication for an infection.</li>
		<li>Ensure that the donor does not have a history of constipation or use laxatives chronically. Additionally, ensure the donor does not have a history of Inflammatory Bowel Disease, chronic diarrhea, microscopic colitis, a personal history of intestinal/colonic malignancy, or a history of gastric bypass/other weight loss surgery.</li>
		<li>Ensure the donor has not recently traveled (within the last three months) to an area where infectious diarrhea is a concern. If they have traveled and experienced diarrhea they should be exluded.</li>
		<li>Ensure that the donor is not taking any immunosuppressive agents and is not on systemic chemotherapy.</li>
		<li>Ensure that the donor does not have metabolic syndrome, autoimmune conditions or atopic diseases.</li>
	</ol>
	</li>
	<li>Obtain written consent from the donor and recipient prior to screening given the sensitive nature of the screening tests. Reassure both parties that test results will not be shared with the other party.</li>
</ol>
4. Screening
NOTE: This screening protocol was adapted for the patients at Brigham and Women&#8217;s Hospital in Massachusetts, U.S.A. Consider consulting the infectious disease department and hospital infection control prior to initiating the FMT protocol to make sure all appropriate screening tests have been considered.
<ol>
	<li>Recipient:
	<ol>
		<li>At the time of the initial clinic visit, send the following laboratory tests for recipient screening (to establish baseline testing for these infectious diseases and to ensure there is no question of transmission in the future).</li>
		<li>Perform Hepatitis A (IgG and IgM), Hepatitis B (HbsAg/Ab and HbcAb), Hepatitis C Ab, HIV-1/2 (Ab and viral load) and Syphilis (TP-IgG) laboratory blood tests.</li>
		<li>Obtain a thorough history of food allergies at this initial assessment.</li>
	</ol>
	</li>
	<li>Donor:
	<ol>
		<li>Coordinate with the microbiology laboratory before screening to ensure formed stool is not discarded.</li>
		<li>Order the below laboratory tests for donor screening if the donor meets eligibility criteria as per section 3.</li>
		<li>Perform Hepatitis A (IgG and IgM), Hepatitis B (HBsAg/Ab and HBcAb), Hepatitis C Ab, HIV-1/2 (Ab and viral load) and Syphilis (TP-IgG) laboratory blood tests.</li>
		<li>Perform C. difficile (by culture), routine stool culture, Giardia antigen, Cryptosporidium antigen and ova and parasites laboratory stool tests.</li>
		<li>Screen the donor no more than 30 days but preferably 1 - 2 weeks prior to the scheduled procedure. 
		NOTE: If the donor develops diarrhea or liquid stool they are not longer eligible for donation.</li>
	</ol>
	</li>
	<li>Counsel the donor on foods to avoid based on the recipient&#8217;s food allergies. The donor should not ingest foods to which the recipient is allergic for the 5 days before the donation.</li>
</ol>
5. Pre-procedure Preparation
<ol>
	<li>Recipient:
	<ol>
		<li>At the initial clinic visit, if the recipient is not already on oral vancomycin, start vancomycin 125 mg every 6 hr for at least seven to ten days prior to the procedure.</li>
		<li>Provide the recipient with the endoscopy center&#8217;s instructions for pre-colonoscopy bowel preparation to be done the day before the scheduled procedure. Recipients should also follow the endoscopy center&#8217;s standard dietary instructions for the week prior to the procedure.</li>
		<li>Instruct the recipient to take their last dose of vancomycin the night before the procedure.</li>
	</ol>
	</li>
	<li>Donor:
	<ol>
		<li>Provide the donor with several stool collection cups. Label toilet inserts (hats) and cups with the recipient&#8217;s label as well as 3 empty specimen containers. Ask the donor to begin stool collection no more than 6 hr prior to processing (time of procedure). 
		NOTE: If the donor provides multiple stool samples, they should be labeled with dates and times. Use the freshest sample (one filled specimen cup will suffice).</li>
		<li>Ensure that the recipient to brings the donor&#8217;s stool sample with them to the procedure.</li>
		<li>Store the stool samples at room temperature. Exclude any unformed stool. Discard any unused stool in a sanitary sewer system (unless saving for research purposes).</li>
	</ol>
	</li>
</ol>
6. Stool Preparation
NOTE: Ensure this procedure is discussed and approved by the hospital&#8217;s infection control department.
<ol>
	<li>Wear personal protective equipment when preparing the stool.</li>
	<li>Prepare stool in the endoscopy room in a designated work area in which there is an eighteen inch splash zone that prevents contamination of other materials.</li>
	<li>Prior to stool preparation, clean the designated area with hospital-approved disinfectant.</li>
	<li>Cover the area with a disposable pad to avoid contamination.</li>
	<li>Transfer the formed stool into a fourteen ounce single speed blender and add 500 cc of normal saline.</li>
	<li>Blend the contents for at least one minute or until it reaches a liquid slurry consistency. Be careful to avoid spillage when removing the lid of the blender.</li>
	<li>Pour the solution through the strainer into a plastic eight quart basin to eliminate any solid waste. Discard the strainer.</li>
	<li>Draw up the strained solution into 60 cc luer lock syringes (approximately eight to nine for a standard FMT by colonoscopy). Draw up an extra 60 cc of normal saline for flushes.</li>
</ol>
7. Fecal Transplant via Colonoscopy
<ol>
	<li>Wear personal protective equipment in accordance with the endoscopy unit policies while performing or assisting with the infusion.</li>
	<li>Consider light sedation, if possible, so that the patient may be able to cooperate with retaining the stool after the procedure.</li>
	<li>Perform a colonoscopy to the terminal ileum (or as far as technically feasible). On insertion of the colonscope, lavage and suction any residual liquid stool.</li>
	<li>Inspect the mucosal walls for evidence of inflammation but mucosal biopsies are not routinely performed during the FMT procedure. 
	NOTE: Determine whether or not a full colonoscopy is safe due to the degree of inflammation. If not,consider a more limited flexible sigmoidoscopy with infusion (use half of the stool slurry).</li>
	<li>Infuse 60 cc (one syringe) once in the terminal ileum via the biopsy channel of the colonoscope using the biopsy channel cap with extension tubing.</li>
	<li>Between applications of stool syringes, flush the channel with normal saline to avoid clogging.</li>
	<li>Infuse another 60 cc of stool into the cecum and then repeat in the right colon.</li>
	<li>Continue infusing 30 cc every five to ten cm while evenly distributing the slurry on all four colon wall quadrants until the mid-transverse colon is reached. Remember to flush with saline in between stool syringes. 
	NOTE: If the slurry is very viscous it may be necessary to push with considerable force on the syringe. Be careful that the biopsy channel cap does not separate from the colonoscope and cause spillage of stool contents. Frequent flushing will allow for easier passage of the slurry. If there is continued difficulty in pushing the slurry, consider re-straining the remaining donor stool for a second time or diluting further with normal saline.</li>
	<li>Aspirate excess air upon withdrawal when changing syringe barrels while being careful not to suction any liquid stool. 
	NOTE: If the patient has known fecal incontinence, the last syringe should be infused no lower than the hepatic flexure to avoid leakage during and post procedure.</li>
	<li>Once the colonoscopy with FMT is complete, advise the patient to retain the stool as long as they can. They may resume their regular diet and medications two hours after the colonoscopy is complete.</li>
</ol>
8. Colonoscopy with FMT Clean Up
<ol>
	<li>Perform post procedure equipment reprocessing, environment cleaning/disinfection and waste disposal as per hospital protocol.</li>
	<li>Dispose of all fecal material in the appropriate biohazard container.</li>
	<li>Bring the colonoscope to the decontamination area for routine processing per standard protocol.</li>
	<li>Dispose of the blender and strainer with any other soiled waste. 
	NOTE: If single use blenders and strainers are not feasible at an individual institution alternate mixing methods can be considered. One example: passing the stool through a stainless steel potato ricer that can be cleaned in an autoclave. We recommend discussing any potential methods with your facility&#8217;s infection control department.</li>
	<li>Decontaminate all surfaces that were used for the procedure using the facility-approved products.</li>
</ol>
9. Follow Up
<ol>
	<li>Arrange a follow up appointment in gastroenterology clinic for the patient approximately 30 days after the FMT.</li>
	<li>Counsel the patient about the risk of C. difficile recurrence with future courses of antibiotics.</li>
</ol>
10. Reporting of Adverse Events
<ol>
	<li>Although FMT is considered to be a safe procedure, it is strongly advised to keep track of all adverse events felt to be associated with the procedure and to report them to the appropriate regulatory boards.</li>
	<li>In order to document the long-term safety of FMT, keep a reliable database that can be periodically queried.</li>
</ol>

<ol>
	<li>Eiseman, B., Silen, W., Bascom, G. S., Kauvar, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fecal+enema+as+an+adjunct+in+the+treatment+of+pseudomembranous+enterocolitis.">Fecal enema as an adjunct in the treatment of pseudomembranous enterocolitis.</a> Surgery. 44 (5), 854-859 (1958).</li><li>Zhang, F., Luo, W., Shi, Y., Fan, Z., Ji, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Should+we+standardize+the+1,700-year-old+fecal+microbiota+transplantation.">Should we standardize the 1,700-year-old fecal microbiota transplantation.</a> Am J Gastroenterol. 107 (11), 1755-1710 (2012).</li><li>Borody, T. J., Paramsothy, S., Agrawal, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fecal+microbiota+transplantation:+indications,+methods,+evidence,+and+future+directions.">Fecal microbiota transplantation: indications, methods, evidence, and future directions.</a> Curr Gastroenterol Rep. 15 (8), 337 (2013).</li><li>Mattila, E., 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=Fecal+transplantation,+through+colonoscopy,+is+effective+therapy+for+recurrent+Clostridium+difficile+infection.">Fecal transplantation, through colonoscopy, is effective therapy for recurrent Clostridium difficile infection.</a> Gastroenterology. 142 (3), 490-496 (2012).</li><li>Brandt, L. J., Aroniadis, O. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+overview+of+fecal+microbiota+transplantation:+techniques,+indications,+and+outcomes.">An overview of fecal microbiota transplantation: techniques, indications, and outcomes.</a> Gastrointest Endosc. 78 (2), 240-249 (2013).</li><li>Aas, J., Gessert, C. E., Bakken, J. 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The authors have nothing to disclose relevant to the material presented in this manuscript.

Fecal transplants have been shown to be an effective and safe treatment for recurrent CDI. We have outlined a standard protocol for FMT via colonoscopy and included our results for 22 patients who underwent the procedure for recurrent CDI. Our results thus far are comparable to published data for recurrent disease with only one patient recurring one month post FMT. A critical step of the procedure is the prescreening visit to ensure that the donor is suitable and that the patient has no contraindications to unde...

Fecal transplants for the treatment of pseumembranous colitis were first described in 19581 . However, reports of ingesting feces for the treatment of food poisoning and severe diarrhea date back as early as fourth century China2 . Fecal microbiota transplantation (FMT) is also termed in the literature &#8220;fecal bacteriotherapy&#8221;, &#8220;human probiotic infusion&#8221;, &#8220;stool transplant&#8221;, &#8220;intestinal microbiome restoration&#8221;, and &#8220;fecal transfer&#8221;. It involves collecting stool from a healthy, pre-screened donor and delivering a prepared slurry into the gastrointestinal tract of the recipient via nasogastric tube, esophagogastricduodenoscopy, colonoscopy, or enema 3 . Many case series have reported the efficacy of administration via NGT and enema, however more recent studies have shown that administration of donor stool via a colonoscopy is safe and highly effective4 . While other methods such as nasogastric tube administration have been reported, delivery of donor stool via colonoscopy has less immediate side effects (e.g. abdominal bloating and nausea)4 and we have found that patients are more accepting of the concept of FMT when it is performed by colonoscopy. One of the therapeutic advantages of FMT via colonoscopy is the reduction in colonic biomass due to the bowel lavage that is performed prior to the procedure4.
Multiple studies have investigated the role of FMT for the treatment of colitis and diarrhea caused by the opportunistic pathogen C. difficile (CDI). The incidence of CDI continues to rise and is a major cause of morbidity and mortality with a large economic burden throughout the world. First line treatment for CDI consists of antibiotic therapy, however recurrence rates have been reported between 15-35%5 . Several case series and reports have documented the safety and efficacy of FMT for CDI refractory to standard medical treatment with antibiotics4,6-16 . A study looking at long term follow up of patients after FMT via colonoscopy for CDI reported a 91% primary cure rate in 77 patients17.
At our Center (Brigham and Women&#39;s Hospital Division of Gastroenterology, Hepatology and Endoscopy), we initiated a fecal transplant program for patients with recurrent or refractory CDI. Appropriate candidates are defined as patients who have recurrent CDI (a history of 3 or more episodes, or 2 episodes that required hospitalization), or patients with refractory disease that is unresponsive to traditional antibiotics. We believe a systematic approach to all phases of this procedure maximizes efficacy. In this manuscript and accompanying video, we detail the protocol we have employed at our Center, which includes patient and donor screening, stool preparation, and the delivery of stool at the time of colonoscopy. This method has yielded positive results comparable to the published literature.
CASE PRESENTATION:
This patient represents a typical patient referred for fecal transplantation.
The patient is a 69 year-old woman with a history of chronic lymphocytic leukemia who had relapse of disease requiring further treatment with chemotherapy. She suffered from three episodes of CDI in the past year, and was therefore referred to our clinic for consideration of FMT prior to re-initiating chemotherapy. Her first episode of CDI occurred in October 2012. She had not had any preceding antibiotics. She was very ill in the ICU after presenting with septic shock and underwent diverting loop ileostomy with antegrade vancomycin enemas for a 6 week course in combination with oral vancomycin given the severity of her illness. She did very well with resolution of her diarrhea and she was able to come off antibiotics. She developed a hernia around her stoma so it was reversed. Shortly after the ostomy reversal she again developed diarrhea and was found again to be C. difficile toxin positive. She completed another 6 week course of oral vancomycin and was able to taper off of it successfully. However several months later, she again developed diarrhea that was C. difficile toxin positive and was started on her third course of oral vancomycin. She was doing well on antibiotics when it was noted that her white count increased to &#62;100 and she has found to have a recurrence of her CLL on bone marrow biopsy. Her oncologists were concerned about initiating chemotherapy without complete resolution of her CDI and so we were asked to perform FMT. &#8206;

<table border="0" cellpadding="0" cellspacing="0" width="840">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Name of Material/ Equipment</td>
			<td style="width:121px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:355px;">Comments/Description</td>
		</tr>
		<tr height="92">
			<td height="92" style="height:92px;width:247px;">Blender</td>
			<td style="width:121px;">Hamilton Beach</td>
			<td style="width:119px;"></td>
			<td style="width:355px;">http://www.amazon.com/Hamilton-Beach-51101-Personal-Blender/dp/B00065L6CU/ref=pd_sim_k_2?ie=UTF8&#38;refRID=1Q06MXAE9XKTB4QG0AEN</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:247px;">Strainer</td>
			<td style="width:121px;">Winco MS3A-8S Strainer</td>
			<td style="width:119px;"></td>
			<td style="width:355px;">http://www.amazon.com/Winco-MS3A-8S-Strainer-Single-Wooden/dp/B001VZ5E40/ref=sr_1_3?s=home-garden&#38;ie=UTF8&#38;qid=1395001295&#38;sr=1-3&#38;keywords=kitchen+strainer+wooden+handle</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Disposable pads (chux), 17in x 24in.&#160;</td>
			<td>hospital standard</td>
			<td style="width:119px;"></td>
			<td>one package&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Pink rectangular hospital standard wash basin, 8 quart&#160;</td>
			<td>hospital standard</td>
			<td style="width:119px;"></td>
			<td>1 or 2</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">500 cc bottle of sterile normal saline</td>
			<td style="width:121px;">hospital standard</td>
			<td style="width:119px;"></td>
			<td>2</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">60cc leur lock syringes</td>
			<td style="width:121px;">hospital standard</td>
			<td style="width:119px;"></td>
			<td>8 or 9</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Biopsy channel cap with extension</td>
			<td style="width:121px;">hospital standard</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

We have performed 24 FMTs on 22 patients at our Center (Table 1) using the above protocol. Nine patients had concurrent IBD (six with Crohn&#8217;s disease and three with ulcerative colitis). All nine had resolution of CDI. One of the patients with Crohn&#8217;s disease was status post a total colectomy for Crohn&#8217;s colitis. He initially underwent FMT via colonoscopy however after experiencing a recurrence two weeks post FMT he underwent a second FMT via upper endoscopy which resulted in CDI resolut...

Watch this Scientific Journal Video about Fecal Microbiota Transplantation via Colonoscopy for Recurrent C. difficile Infection at JoVE.com

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 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 ...

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Research

JoVE Journal

Medicine

27.0K Views.  Brigham and Women‘s Hospital. 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.

Video: Fecal Microbiota Transplantation via Colonoscopy for Recurrent C. difficile Infection

A Mouse Model of in Utero Transplantation

The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus. For example, in utero transplantation (IUT) of hematopoietic stem cells has been used to successfully treat patients with severe combined immunodeficiency.1,2 In several other conditions, however, IUT has been attempted without success.3 Given these mixed results, the availability of an efficient non-human model to study the biological sequelae of stem cell transplantation and gene therapy is critical to advance this field. We and others have used the mouse model of IUT to study factors affecting successful engraftment of in utero transplanted hematopoietic stem cells in both wild-type mice4-7 and those with genetic diseases.8,9 The fetal environment also offers considerable advantages for the success of in utero gene therapy. For example, the delivery of adenoviral10, adeno-associated viral10, retroviral11, and lentiviral vectors12,13 into the fetus has resulted in the transduction of multiple organs distant from the site of injection with long-term gene expression. in utero gene therapy may therefore be considered as a possible treatment strategy for single gene disorders such as muscular dystrophy or cystic fibrosis. Another potential advantage of IUT is the ability to induce immune tolerance to a specific antigen. As seen in mice with hemophilia, the introduction of Factor IX early in development results in tolerance to this protein.14 
In addition to its use in investigating potential human therapies, the mouse model of IUT can be a powerful tool to study basic questions in developmental and stem cell biology. For example, one can deliver various small molecules to induce or inhibit specific gene expression at defined gestational stages and manipulate developmental pathways. The impact of these alterations can be assessed at various timepoints after the initial transplantation. Furthermore, one can transplant pluripotent or lineage specific progenitor cells into the fetal environment to study stem cell differentiation in a non-irradiated and unperturbed host environment.
The mouse model of IUT has already provided numerous insights within the fields of immunology, and developmental and stem cell biology. In this video-based protocol, we describe a step-by-step approach to performing IUT in mouse fetuses and outline the critical steps and potential pitfalls of this technique.

A Mouse Model of in Utero Transplantation

The mouse model of in utero transplantation is a versatile tool that can be used to study the potential clinical applications of stem cell transplantation and gene therapy in the fetus. In this protocol, we present a general approach to performing this technique

The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus.  For example, in ...

Ex Vivo Infection of Live Tissue with Oncolytic Viruses

Oncolytic Viruses (OVs) are novel therapeutics that selectively replicate in and kill tumor cells1. Several clinical trials evaluating the effectiveness of a variety of oncolytic platforms including HSV, Reovirus, and Vaccinia OVs as treatment for cancer are currently underway2-5. One key characteristic of oncolytic viruses is that they can be genetically modified to express reporter transgenes which makes it possible to visualize the infection of tissues by microscopy or bio-luminescence imaging6,7. This offers a unique advantage since it is possible to infect tissues from patients ex vivo prior to therapy in order to ascertain the likelihood of successful oncolytic virotherapy8. To this end, it is critical to appropriately sample tissue to compensate for tissue heterogeneity and assess tissue viability, particularly prior to infection9. It is also important to follow viral replication using reporter transgenes if expressed by the oncolytic platform as well as by direct titration of tissues following homogenization in order to discriminate between abortive and productive infection. The object of this protocol is to address these issues and herein describes 1. The sampling and preparation of tumor tissue for cell culture 2. The assessment of tissue viability using the metabolic dye alamar blue 3. Ex vivo infection of cultured tissues with vaccinia virus expressing either GFP or firefly luciferase 4. Detection of transgene expression by fluorescence microscopy or using an In Vivo Imaging System (IVIS) 5. Quantification of virus by plaque assay. This comprehensive method presents several advantages including ease of tissue processing, compensation for tissue heterogeneity, control of tissue viability, and discrimination between abortive infection and bone fide viral replication.

Ex Vivo Infection of Live Tissue with Oncolytic Viruses

Oncolytic viruses are promising for cancer therapeutics. The ability to ascertain the infectability of live tissue specimens obtained from patients prior to treatment is a unique advantage of this therapeutic approach. This protocol describes how to process tissues for ex vivo infection with oncolytic virus and subsequent viral quantification.

Oncolytic Viruses (OVs) are novel therapeutics that selectively replicate in and kill tumor cells1. Several clinical trials evaluating the effectiveness ...

Intraspinal Cell Transplantation for Targeting Cervical Ventral Horn in Amyotrophic Lateral Sclerosis and Traumatic Spinal Cord Injury

Respiratory compromise due to phrenic motor neuron loss is a debilitating consequence of a large proportion of human traumatic spinal cord injury (SCI) cases 1 and is the ultimate cause of death in patients with the motor neuron disorder, amyotrophic laterals sclerosis (ALS) 2.
ALS is a devastating neurological disorder that is characterized by relatively rapid degeneration of upper and lower motor neurons. Patients ultimately succumb to the disease on average 2-5 years following diagnosis because of respiratory paralysis due to loss of phrenic motor neuron innnervation of the diaphragm 3. The vast majority of cases are sporadic, while 10% are of the familial form. Approximately twenty percent of familial cases are linked to various point mutations in the Cu/Zn superoxide dismutase 1 (SOD1) gene on chromosome 21 4. Transgenic mice 4,5 and rats 6 carrying mutant human SOD1 genes (G93A, G37R, G86R, G85R) have been generated, and, despite the existence of other animal models of motor neuron loss, are currently the most highly used models of the disease.
Spinal cord injury (SCI) is a heterogeneous set of conditions resulting from physical trauma to the spinal cord, with functional outcome varying according to the type, location and severity of the injury 7. Nevertheless, approximately half of human SCI cases affect cervical regions, resulting in debilitating respiratory dysfunction due to phrenic motor neuron loss and injury to descending bulbospinal respiratory axons 1. A number of animal models of SCI have been developed, with the most commonly used and clinically-relevant being the contusion 8.
Transplantation of various classes of neural precursor cells (NPCs) is a promising therapeutic strategy for treatment of traumatic CNS injuries and neurodegeneration, including ALS and SCI, because of the ability to replace lost or dysfunctional CNS cell types, provide neuroprotection, and deliver gene factors of interest 9.
Animal models of both ALS and SCI can model many clinically-relevant aspects of these diseases, including phrenic motor neuron loss and consequent respiratory compromise 10,11. In order to evaluate the efficacy of NPC-based strategies on respiratory function in these animal models of ALS and SCI, cellular interventions must be specifically directed to regions containing therapeutically relevant targets such as phrenic motor neurons. We provide a detailed protocol for multi-segmental, intraspinal transplantation of NPCs into the cervical spinal cord ventral gray matter of neurodegenerative models such as SOD1G93A mice and rats, as well as spinal cord injured rats and mice 11.

Neural precursor transplantation is a promising strategy for protecting and/or replacing lost/dysfunctional cervical phrenic motor neurons in spinal cord injury (SCI) and the motor neuron disorder, amyotrophic laterals sclerosis (ALS). We provide a protocol for cell delivery to cervical spinal cord ventral horn in rodent models of ALS and SCI.

Respiratory compromise due to phrenic motor neuron loss is a debilitating consequence of a large proportion of human traumatic spinal cord injury (SCI) ...

Orthotopic Small Bowel Transplantation in Rats

Small bowel transplantation has become an accepted clinical option for patients with short gut syndrome and failure of parenteral nutrition (irreversible intestinal failure). In specialized centers improved operative and managing strategies have led to excellent short- and intermediate term patient and graft survival while providing high quality of life 1,3. Unlike in the more common transplantation of other solid organs (i.e. heart, liver) many underlying mechanisms of graft function and immunologic alterations induced by intestinal transplantation are not entirely known6,7. Episodes of acute rejection, sepsis and chronic graft failure are the main obstacles still contributing to less favorable long term outcome and hindering a more widespread employment of the procedure despite a growing number of patients on home parenteral nutrition who would potentially benefit from such a transplant. The small intestine contains a large number of passenger leucocytes commonly referred to as part of the gut associated lymphoid system (GALT) this being part of the reason for the high immunogenity of the intestinal graft. The presence and close proximity of many commensals and pathogens in the gut explains the severity of sepsis episodes once graft mucosal integrity is compromised (for example by rejection). To advance the field of intestinal- and multiorgan transplantation more data generated from reliable and feasible animal models is needed. The model provided herein combines both reliability and feasibility once established in a standardized manner and can provide valuable insight in the underlying complex molecular, cellular and functional mechanisms that are triggered by intestinal transplantation. We have successfully used and refined the described procedure over more than 5 years in our laboratory 8-11. The JoVE video-based format is especially useful to demonstrate the complex procedure and avoid initial pitfalls for groups planning to establish an orthotopic rodent model investigating intestinal transplantation.

Small bowel transplantation has become an accepted treatment option for patients with irreversible intestinal failure. Our experimental model of orthotopic small bowel transplantation in rats serves as a reliable tool to address underlying immunologic and inflammatory processes that complicate intestinal transplantation.

Small  bowel transplantation has become an accepted clinical option for patients with  short gut syndrome and failure of parenteral nutrition ...

Bronchoalveolar Lavage (BAL) for Research; Obtaining Adequate Sample Yield

We describe a research technique for fiberoptic bronchoscopy with bronchoalveolar lavage (BAL) using manual hand held suction in order to remove nonadherent cells and lung lining fluid from the mucosal surface. In research environments, BAL allows sampling of innate (lung macrophage), cellular (B- and T- cells), and humoral (immunoglobulin) responses within the lung.
BAL is internationally accepted for research purposes and since 1999 the technique has been performed in &#62; 1,000 subjects in the UK and Malawi by our group.
Our technique uses gentle hand-held suction of instilled fluid; this is designed to maximize BAL volume returned and apply minimum shear force on ciliated epithelia in order to preserve the structure and function of cells within the BAL fluid and to preserve viability to facilitate the growth of cells in ex vivo culture. The research technique therefore uses a larger volume instillate (typically in the order of 200 ml) and employs manual suction to reduce cell damage.
Patients are given local anesthetic, offered conscious sedation (midazolam), and tolerate the procedure well with minimal side effects. Verbal and written subject information improves tolerance and written informed consent is mandatory. Safety of the subject is paramount. Subjects are carefully selected using clear inclusion and exclusion criteria.
This protocol includes a description of the potential risks, and the steps taken to mitigate them, a list of contraindications, pre- and post-procedure checks, as well as precise bronchoscopy and laboratory techniques.

Bronchoalveolar Lavage BAL for Research; Obtaining Adequate Sample Yield

We describe a research technique for fiberoptic bronchoscopy and bronchoalveolar lavage using low pressure suction. The technique is used to harvest immune cells from the lung bronchoalveolar surfaces. Local anesthetic and mild conscious sedation (midazolam) is used. Subjects tolerate the procedure well and experience minimal side effects.

We describe a research technique for fiberoptic bronchoscopy with bronchoalveolar lavage (BAL) using manual hand held suction in order to remove ...

Tumor Treating Field Therapy in Combination with Bevacizumab for the Treatment of Recurrent Glioblastoma

A novel device that employs TTF therapy has recently been developed and is currently in use for the treatment of recurrent glioblastoma (rGBM). It was FDA approved in April 2011 for the treatment of patients 22 years or older with rGBM. The device delivers alternating electric fields and is programmed to ensure maximal tumor cell kill1.
Glioblastoma is the most common type of glioma and has an estimated incidence of approximately 10,000 new cases per year in the United States alone2. This tumor is particularly resistant to treatment and is uniformly fatal especially in the recurrent setting3-5. Prior to the approval of the TTF System, the only FDA approved treatment for rGBM was bevacizumab6. Bevacizumab is a humanized monoclonal antibody targeted against the vascular endothelial growth factor (VEGF) protein that drives tumor angiogenesis7. By blocking the VEGF pathway, bevacizumab can result in a significant radiographic response (pseudoresponse), improve progression free survival and reduce corticosteroid requirements in rGBM patients8,9. Bevacizumab however failed to prolong overall survival in a recent phase III trial26. A pivotal phase III trial (EF-11) demonstrated comparable overall survival between physicians&#8217; choice chemotherapy and TTF Therapy but better quality of life were observed in the TTF arm10.
There is currently an unmet need to develop novel approaches designed to prolong overall survival and/or improve quality of life in this unfortunate patient population. One appealing approach would be to combine the two currently approved treatment modalities namely bevacizumab and TTF Therapy. These two treatments are currently approved as monotherapy11,12, but their combination has never been evaluated in a clinical trial. We have developed an approach for combining those two treatment modalities and treated 2 rGBM patients. Here we describe a detailed methodology outlining this novel treatment protocol and present representative data from one of the treated patients.

A novel methodology that is employed for the treatment of recurrent glioblastomas is described. This treatment approach employs the application of alternating electric tumor treating fields (TTFields), known as TTF Therapy in combination with bevacizumab, a targeted agent that is currently FDA approved as monotherapy.

A novel device that employs TTF therapy has recently been developed and is currently in use for the treatment of recurrent glioblastoma (rGBM). It was FDA ...

Ex Situ Normothermic Machine Perfusion of Donor Livers

In contrast to conventional static cold preservation (0-4 &#176;C), ex situ machine perfusion may provide better preservation of donor livers. Continuous perfusion of organs provides the opportunity to improve organ quality and allows ex situ viability assessment of donor livers prior to transplantation. This video article provides a step by step protocol for ex situ normothermic machine perfusion (37 &#176;C) of human donor livers using a device that provides a pressure and temperature controlled pulsatile perfusion of the hepatic artery and continuous perfusion of the portal vein. The perfusion fluid is oxygenated by two hollow fiber membrane oxygenators and the temperature can be regulated between 10 &#176;C and 37 &#176;C. During perfusion, the metabolic activity of the liver as well as the degree of injury can be assessed by biochemical analysis of samples taken from the perfusion fluid. Machine perfusion is a very promising tool to increase the number of livers that are suitable for transplantation.

Ex Situ Normothermic Machine Perfusion of Donor Livers

Here we present a protocol describing oxygenated ex situ machine perfusion of donor liver grafts. This article contains a step by step protocol to procure and prepare the liver graft for machine perfusion, prepare the perfusion fluid, prime the perfusion machine and perform oxygenated normothermic machine perfusion of the liver graft.

In contrast to conventional static cold preservation (0-4 &#176;C), ex situ machine perfusion may provide better preservation of donor livers. Continuous ...

Veno-Venous Extracorporeal Membrane Oxygenation in a Mouse

The use of extracorporeal membrane oxygenation (ECMO) has increased substantially in recent years. ECMO has become a reliable and effective therapy for acute as well as end-stage lung diseases. With the increase in clinical demand and prolonged use of ECMO, procedural optimization and prevention of multi-organ damage are of critical importance. The aim of this protocol is to present a detailed technique of veno-venous ECMO in a non-intubated, spontaneously breathing mouse. This protocol demonstrates the technical design of the ECMO and surgical steps. This murine ECMO model will facilitate the study of pathophysiology related to ECMO (e.g., inflammation,bleeding and thromboembolic events). Due to the abundance of genetically modified mice, the molecular mechanisms involved in ECMO-related complications can also be dissected.

Here we present a protocol describing the technique of veno-venous extracorporeal membrane oxygenation (ECMO) in a non-intubated, spontaneously breathing mouse. This murine model of ECMO can be effectively implemented in experimental studies of acute and end-stage lung diseases.

The use of extracorporeal membrane oxygenation (ECMO) has increased substantially in recent years. ECMO has become a reliable and effective therapy for ...

Immunofluorescence Labelling of Human and Murine Neutrophil Extracellular Traps in Paraffin-Embedded Tissue

Neutrophil granulocytes, also called polymorphonuclear leukocytes (PMN) due to their lobulated nucleus, are the most abundant type of leukocytes. They mature in the bone marrow and are released into the peripheral blood, where they circulate for about 6&#8722;8 h; however, in tissue, they can survive for days. By diapedesis through the endothelium, they leave the blood stream, enter tissues, and migrate towards the site of an infection following chemotactic gradients.&#160;Neutrophils can combat invading microorganisms by phagocytosis,&#160;degranulation, and generation of neutrophil extracellular traps (NETs). This protocol will help to detect NETs in paraffin-embedded tissue. NETs are the result of a process called NETosis, which leads to the release of nuclear, granular, and cytoplasmic components either from living (vital NETosis) or dying (suicidal NETosis) neutrophils. In vitro, NETs form cloud-like structures, which occupy a space several times larger than that of the cells from which they descended. The backbone of NETs is chromatin, to which a selection of proteins and peptides originating from granules and cytoplasm are bound. Thereby, a high local concentration of toxic compounds is maintained so that NETs can capture and inactivate a variety of pathogens including bacteria, fungi, viruses, and parasites, while diffusion of the highly active NET components leading to damage in neighboring tissue is limited. Nevertheless, in recent years it has become apparent that NETs, if generated in abundance or cleared insufficiently, do have pathological potential ranging from autoimmune diseases to cancer. Thus, detection of NETs in tissue samples may have diagnostic significance, and the detection of NETs in diseased tissue can influence the treatment of patients. Since paraffin-embedded tissue samples are the standard specimen used for pathological analysis, it was sought to establish a protocol for fluorescent staining of NET components in paraffin-embedded tissue using commercially available antibodies.

Neutrophil extracellular traps (NETs) are three-dimensional structures generated by stimulated neutrophil granulocytes. It has become clear in recent years that NETs are involved in a wide variety of diseases. Detection of NETs in tissue may have diagnostic relevance, so standardized protocols for labelling NET components are required.

Neutrophil granulocytes, also called polymorphonuclear leukocytes (PMN) due to their lobulated nucleus, are the most abundant type of leukocytes. They ...

Inguinal Subcutaneous White Adipose Tissue (ISWAT) Transplantation Model of Murine Islets

Pancreatic islet transplantation is a well-established therapeutic treatment for type 1 diabetes. The kidney capsule is the most commonly used site for islet transplantation in rodent models. However, the tight kidney capsule limits the transplantation of sufficient islets in large animals and humans. The inguinal subcutaneous white adipose tissue (ISWAT), a new subcutaneous space, was found to be a potentially valuable site for islet transplantation. This site has better blood supply than other subcutaneous spaces. Moreover, the ISWAT accommodates a larger islet mass than the kidney capsule, and transplantation into it is simple. This manuscript describes the procedure of mouse islet isolation and transplantation in the ISWAT site of syngeneic diabetic mouse recipients. Using this protocol, murine pancreatic islets were isolated by standard collagenase digestion and a basement membrane matrix hydrogel was used for fixing the purified islets in the ISWAT site. The blood glucose levels of the recipient mice were monitored for more than 100 days. Islet grafts were retrieved at day 100 after transplantation for histological analysis. The protocol for islet transplantation in the ISWAT site described in this manuscript is simple and effective.

Inguinal Subcutaneous White Adipose Tissue ISWAT Transplantation Model of Murine Islets

In this protocol, a method of murine islet isolation and transplantation into the inguinal subcutaneous white adipose tissue is described. Isolated syngeneic murine islets are transplanted into a murine recipient using a basement membrane hydrogel. The blood glucose level of the recipients is monitored, and histology analysis of the islet grafts is performed.

Pancreatic islet transplantation is a well-established therapeutic treatment for type 1 diabetes. The kidney capsule is the most commonly used site for ...