During the past eight years, the applicant's laboratory has made many important observations to suggest that the small intestine of human infants/prematures is developmentally immature in its host defenses. These host defense immaturities may account for the increased incidence of gastrointestinal diseases occurring in young infants such as necrotizing enterocolitis, invasive bacterial gastroenteritis, toxigenic diarrhea, and intestinal food allergy. Preliminary developmental biology studies of primary enterocyte (non-immortal) cultures and of intestinal cancer cell lines suggest that the composition of the cell membrane of immature enterocytes facilities pathologic bacterial colonization in the gut, a first step in invasive bacterial gastroenteritis, and the cAMP signal transduction system which controls fluid secretion after toxin interaction with enterocyte receptors is increased in sensitivity to bacterial toxins, accounting for the increase incidence of toxigenic diarrheas in infancy. Furthermore, an increased enterocyte membrane fluidity due to an altered lipid/protein composition in immature animals amy explain the increased transport of intestinal antigens into the infant's circulation leading to intestinal allergic/inflammatory states. In order to more clearly understand enterocyte immaturities in neonates/prematures, cellular/molecular studies on viable human small intestinal enterocytes are needed. Data from these cell biology studies can be used to plan strategies of preventing these neonatal intestinal diseases in humans using breast milk, orally-administered growth factors, etc. Accordingly, this fellowship application proposes to establish an immortal human fetal small intestinal cell line which functions like a normal immature crypt cell and has the capacity to differentiate into mature villus enterocytes. This will be done using primary cultures of 18-20 week fetal small intestinal epithelial cells transfected with viral DNA oncogenes known to transform human epithelial cells. These studies require a laboratory which has developed intestinal cell lines, transformed epithelial cells, can prepare the appropriate plasmids (DNA constructs) which both immortalize epithelial cells, but also allow them to differentiate. The molecular biology and cell biology techniques required to accomplish this goal required a daily input of a gastrointestinal cell biologist such as Dr. Daniel Louvard, Director of the Department of the Molecular Biology and Membrane Biology Group at the Pasteur Institute, Paris, France. He is the leading expert in this research and wishes to establish a mutually beneficial relationship with Dr. Walker's Mucosal Immunology Laboratory at Massachusetts General Hospital. The project will be done over three years, requiring three separate visits to the Pasteur Institute. During the first visit (June 1, 1992- August 31, 1992), the primary human cell cultures will be transfected and clones isolated and characterized. Learning the techniques and nuances of culture conditions as well as approaches to characterizing clones will take three months. Dr. Walker will then further study these cells with conventional techniques in Boston and return to the Pasteur Institute for a second visit (June 1, 1993 - October 15, 1993). At this time he will make additional constructs of viral DNA oncogenes and various promoter genes (villin, alkaline phosphatase, etc.) and try temperature sensitive mutants of SV40 viral oncogenes which may improve the likelihood of establishing a human cell line which differentiates. These experiments are very complex and will require day-to-day supervision on Dr. Louvard's Laboratory. He and his co-workers are uniquely qualified to do these experiments. These clones will then be studied in Boston. Finally, during the third visit (June 1, 1994 - October 15, 1994) the applicant will need help from Dr. Louvard to alter culture conditions or modify transfections of primary cells to establish immortal human cells which not only differentiate, but express the membrane characteristics which he plans to study, i.e. surface glycoconjugates that allow increased bacterial colonization, immature cAMP signal transduction, and altered membrane fluidity. These modifications are extremely difficult and require daily access to the Louvard Laboratory for the 4 1/2 months requested. It i anticipated that this project will be an important component of the ongoing NIH-funded research for the next 5-7 years in the Mucosal Immunology Laboratory at Massachusetts General Hospital-East.
| Sanderson, I R; Ezzell, R M; Kedinger, M et al. (1996) Human fetal enterocytes in vitro: modulation of the phenotype by extracellular matrix. Proc Natl Acad Sci U S A 93:7717-22 |
| Sanderson, I R; Walker, W A (1995) Establishment of a human fetal small intestinal epithelial cell line. Int Arch Allergy Immunol 107:396-7 |
