For these reasons immunohistochemistry of stored paraffin-embedded tissue samples was the only practical method for this initial study. A second limitation is that 18 tissue samples (31%) originated from NEC patients. CD4+or FOXP3+to CD8+ratios, suggesting intact ontogeny of Treg in intestinal tissue early in gestation. == Conclusion == Human infants exhibit presence of mucosal FOXP3+cells in the small and large intestinal mucosa at birth and as early as 23 weeks GA. The frequency of FOXP3+cells and the ratios of FOXP3+to CD4+or CD8+cells do not switch with increasing intrauterine development or postnatal age. Keywords:Enterocolitis, necrotizing, FOXP3 protein, human, immunity, mucosal, immunohistochemistry, infant development, T-lymphocyte == Introduction == Forkhead box protein 3 (FOXP3) is usually a transcription factor predominantly expressed in IL-2R (CD25)highCD4+T cells and its expression is required for regulatory T lymphocyte (Treg) generation and maintenance. Even though validity of FOXP3 to define human Treg is usually debated (1), OI4 currently it remains the most commonly used and unambiguous marker available to identify Treg in mice and humans (2-5). Treg suppress innate and adaptive immune responses, and abnormalities in figures and/or function of these cells have been associated with autoimmune diseases, chronic infections and malignancy (6). In patients with X-chromosome linked mutations of FOXP3 (IPEX syndrome), early development of multi-organ autoimmune diseases, including enteropathy much like inflammatory bowel syndrome, ensues (7). Intestinal immune homeostasis despite exposure to billions of bacteria is considered a unique feature of the mature human intestinal tract. The intestinal tract of the premature infant is prone to inflammatory complications such as necrotizing enterocolits (NEC) (8). It is therefore important to determine the postnatal ontogeny of intestinal Treg because relative lack of these cells can lead to inflammatory enteropathy (9) and their ontogeny may explain why premature infants are at higher risk for NEC. While Treg were first described as differentiating in the newborn thymus (10), Treg can also be converted in the periphery from non-Treg cells (11). Treg can be detected in the human fetal thymus at 13 weeks of gestation, approximately 4 weeks after the first appearance of adaptive T and B cells (12), suggesting an important role in tolerance to maternal antigens (13). Treg enter the fetal lymph nodes, spleen, liver and bone marrow by 14 to 17 weeks, where they acquire a primed/memory phenotype and are fully functional (13,14). Treg are abundant in cord blood and represent 15% of CD4+T cells at 23 weeks and 7% at term, indicating a negative correlation with gestational age (15). Treg appear to be especially important in development in the early postnatal phase when proliferation of self-reactive T cells may be increased during a period of relative lymphopenia (14,16). In contrast, no data is usually available on the presence or ontogeny of fetal intestinal Treg. The gastrointestinal tract is the largest immunological organ of the human body, and immune-mediated gastrointestinal disorders such as inflammatory bowel Mecamylamine Hydrochloride disease (IBD) have been associated with a relative lack of Treg, compared with non-IBD inflammatory controls (17-20). In preterm infants NEC is usually a severe, multifactorial disease of mainly the small intestinal tract characterized by dysregulated immunological and inflammatory host responses (7,21). Lower figures or poor function of Treg may play a role in the NEC disease process. During fetal development, there is a linear increase in Mecamylamine Hydrochloride the density of lamina propria CD3+cells between 12 and 16 weeks gestational age (GA) (22). After 11 weeks GA, the majority of T cells in the lamina propria are CD4+, whereas epithelial T cells are CD8+(23). Culturing of fetal human small intestine with pokeweed mitogen resulted in high numbers of activated T cells and macrophages causing mucosal destruction (24). However in contrast to effector cells, the ontogeny of Treg in the human gastrointestinal tract remains unknown to date. It is conceivable that with increasing exposure to microbiota after birth intestinal Treg figures also rise. Thus, the objective of Mecamylamine Hydrochloride this study was to define the postnatal developmental regulation of small and large intestinal Treg in preterm and term infants in relationship to other T cells. == MATERIALS AND METHODS == == Tissue samples == Remnant human tissue samples not needed for diagnosis are made available for research purposes through the Vanderbilt Human Tissue Acquisition and Pathology Shared Resource (VICC). Between the years 2003 and 2006, we identified.