In previous studies exploring the intestinal epithelium as a potential site for somatic gene therapy, we concluded that the mucus lining the intestine constitutes a significant barrier to any attempts at gene transfer via the lumenal route. The mucus problem is aggravated by the fact that the epithelial stem cells, which are the logical target for gene transfer, are located deep in the intestinal crypts. The goals of the current study were to develop procedures that would improve accessibility to the intestinal stem cells and which would effect in vivo mucus removal without damaging the underlying epithelium. Initial experiments involved evaluation of the use of distension to improve accessibility to the intestinal crypts and the use of the mucolytic agents dithiothreitol (DTT) and N-acetyl-cysteine (NAC) versus a control solution of phosphate-buffered saline (PBS) for mucus removal. Catheters were inserted in each end of 3-cm terminal ileal segments in anesthetized rats. Two milliliters of agent was instilled into the clamped segment for 2 min, removed, and repeated. Lumenal distension resulted in shortened villi with wider intervillus spacing, thereby improving crypt access. Both NAC and DTT washes removed significant mucus between the villi but failed to reach the crypt lumen. To enhance mucus release from the crypt lumen, pilocarpine was selected due to its cholinergic properties and preferential binding to muscarinic receptors on crypt goblet cells. Pilocarpine given intraperitoneally 30 min prior to the mucolytic or PBS wash resulted in significant eradication of mucus down into the crypt lumen. This effect was still evident 3-4 hr later provided the intestine remained undisturbed.

To assess the fetal intestine as a site for gene therapy, we have explored a xenograft model in which fetal rat intestine is grafted subcutaneously into nu/nu mice. Prior to grafting, the tissue was exposed to a replication-deficient retroviral vector bearing the neo gene. Transduction efficiency was assessed by quantitative polymerase chain reaction (PCR) of neo in DNA recovered from the grafts. Three methods of infection were employed: (i) simple flushing of the fetal intestine with the vector; (ii) incubation with the vector for 2 hr; and (iii) a combination of both. The first method gave the highest transduction efficiencies in terms of both the proportion of samples that were neo-positive and the number of neo-positive cells per sample. Using this approach, the time course of persistence of neo-positive cells was analyzed by collecting grafts at 1 versus 3 weeks post-infection. The results showed approximately five-fold more positive cells at the earlier time point than at the later, suggesting loss of transduced cells due to cell turnover. Nevertheless, the persistence of a portion of the positive cells for at least 3 weeks is encouraging for future studies with fetal intestine.

Previous studies in rodent models have demonstrated the feasibility of gene transfer to the stem cells of the intestinal epithelium using ecotropic retroviral vectors delivered luminally. This report represents a next step toward targeting the human intestine as a site for somatic gene therapy. The first experiment assessed the viability of amphotropic retroviral vectors in the luminal environment. It was found that after 4 hr at 37 degrees C in luminal effluent, the loss of titer was no greater than when incubated in control media. Likewise, neither the vector nor the target cells were adversely affected by N-acetylcysteine, which is likely to be used as a preparatory agent for mucus removal. To determine whether human intestinal cells are transducible by these vectors, three colon carcinoma cell lines were studied: HT-29, T84, and Caco-2. All were transduced; however, the expression of the reporter gene was highest in the HT-29 cells. Subsequent studies using these cells showed that with regular stocks of vector, gene transfer peaked at a stock dilution of 1/10 and declined at full strength. This problem could be partially overcome by centrifugal concentration of the retroviral stocks. With this approach, gene transfer increased with increasing particles up to 10x regular stock titers but was inefficient at 100x. Overall, these findings provide encouraging evidence that amphotropic retroviral vectors may eventually be used for in vivo gene transfer into human intestinal epithelium. However, they also point to the need for improved methods of concentrating retroviral vectors.

No abstract

Introduction: Many medical students' initial experience obtaining a history from a pediatric patient happens in their clerkship years. There is a shift in medical education to provide early clinical experiences to train physicians. To increase the exposure to pediatric history in the pre-clinical years, we developed this simulation-based session involving students in their second year of medical school. They are tasked with eliciting a history from a baby provided by a teenager who functions both as a standardized patient (SP) and the parent of the infant. Our goal was to have second-year medical students learn and practice interviewing an adolescent while obtaining history about an infant to assist in the transition to Year three Pediatric clerkship.Approach: Collaborating with the Office of Diversity at our medical school, we recruited students registered in medical academies in public middle and high schools in our county and asked them to be part of this simulation-based activity. A majority of these medical academy students are underrepresented in medicine (URiM). The students functioned as SPs for pre-clerkship medical students while gaining exposure to a career in medicine and the medical school environment. The medical students obtained a history, with faculty providing formative feedback, followed by documentation of the encounter.Outcomes: Medical students felt they gained skills to communicate with caregivers of pediatric patients. They also practiced the skill of eliciting a pediatric history from an infant whose parent is a teenager. The middle and high school students that functioned as SPs gained a better appreciation for the medical education system and felt that the experience was valuable for all parties involved. Discussion: This session exposed pre-clerkship medical students to the nuances of eliciting a pediatric history from pediatric caregivers while also engaging URiM from middle and high school in the medical education process. This session could be used at other institutions to expand diversity in the medical field while also providing pre-clerkship medical students with pediatric experiences.