13 research outputs found
High-throughput gene discovery in the rat
The rat is an important animal model for human diseases and is widely used in physiology. In this article we present a new strategy for gene discovery based on the production of ESTs from serially subtracted and normalized cDNA libraries, and we describe its application for the development of a comprehensive nonredundant collection of rat ESTs. Our new strategy appears to yield substantially more EST clusters per ESTs sequenced than do previous approaches that did not use serial subtraction. However, multiple rounds of library subtraction resulted in high frequencies of otherwise rare internally primed cDNAs, defining the limits of this powerful approach. To date, we have generated >200,000 3′ ESTs from >100 cDNA libraries representing a wide range of tissues and developmental stages of the laboratory rat. Most importantly, we have contributed to ∼50,000 rat UniGene clusters. We have identified, arrayed, and derived 5′ ESTs from >30,000 unique rat cDNA clones. Complete information, including radiation hybrid mapping data, is also maintained locally at http://genome.uiowa.edu/clcg.html. All of the sequences described in this article have been submitted to the dbEST division of the NCBI
Su1938 Lactobacillus Reuteri DSM 17938 Increases FOXP3+ Regulatory T Cells in the Intestine of Rats With Necrotizing Enterocolitis
<em>Lactobacillus reuteri</em> DSM 17938 Changes the Frequency of Foxp3<sup>+</sup> Regulatory T Cells in the Intestine and Mesenteric Lymph Node in Experimental Necrotizing Enterocolitis
<div><p>Necrotizing enterocolitis (NEC) is an inflammatory disease of the intestine in premature infants. <i>Lactobacillus reuteri</i> DSM 17938 improves survival and reduces the incidence and severity of NEC in a rodent model. Foxp3<sup>+</sup> regulatory T cells (Tregs) maintain intestinal homeostasis by controlling inflammation and inducing tolerance. To determine whether there are insufficient numbers of Tregs to control inflammation in NEC and to determine if LR17938 increases the frequency of Tregs, we studied selected groups of newborn Sprague-Dawley rats according to feeding plan: dam±LR17938, formula±LR17938, and NEC±LR17938. NEC was induced by gavage feeding with special formula and exposure to hypoxic conditions. Lymphocytes isolated from ileum, mesenteric lymph nodes (MLN), spleen and thymus were labeled for T cell surface markers (CD3, CD4, CD8) and intracellular Foxp3; and labeled cells were analyzed by flow cytometry. The percentage of CD3<sup>+</sup> T cells and Foxp3<sup>+</sup> Tregs in the ileum significantly decreased in pups with NEC, compared to normal controls. Feeding LR17938 to neonatal rats with NEC increased the % of Foxp3<sup>+</sup> T cells in the ileum while decreasing the percentage of cells in the MLN. Administration of LR17938 to dam-fed rats significantly increased Foxp3<sup>+</sup>Tregs in the ileum as early as day of life (DOL)1 but did not produce an increase in Tregs in formula-fed rats on DOL1. These results suggest that factors in breast milk may enhance the early immunomodulatory effects of LR17938. An anti-inflammatory effect of LR17938 in NEC was associated with the modulation of immune responses and induction and what appears to be migration of Foxp3<sup>+</sup> Tregs to the diseased gut. Probiotic-facilitated development of Tregs might play an important role in the prevention of NEC.</p> </div
Phenotypic composition of T cell subsets in the ileum and mesenteric lymph nodes (MLN) of normal rats during day of life (DOL) 0 to DOL 4.
<p>The frequencies of T cell subsets including CD3<sup>+</sup>, CD4<sup>+</sup>, CD8<sup>+</sup>, CD4<sup>+</sup>CD8<sup>+</sup>, CD4<sup>+</sup>Foxp3<sup>+</sup>, and double positive CD4<sup>+</sup>CD8<sup>+</sup>Foxp3<sup>+</sup> cells, and the comparisons between DOL 0 and DOL 4 were shown by representative flow cytometry plots in ileum (1A–a) and mesenteric lymph node (1B–a) of newborn rats. The bars reflect the percentage of cells, shown as means ± SE, N = 9 pups at each DOL. Comparisons were made between on DOLs 1, 2, 3, or 4 compared with DOL0 in the ileum (1A–b) or MLN (1B–b) of newborn rats, respectively. *p<0.05, **p<0.01, and ***p<0.001.</p
FoxP3<sup>+</sup> Regulatory T Cells Attenuate Experimental Necrotizing Enterocolitis
<div><p>Necrotizing enterocolitis (NEC) results from severe intestinal inflammation in premature infants. FoxP3<sup>+</sup> regulatory T cells (Tregs) are central to gut homeostasis. While Treg proportions are significantly reduced in the ileums of premature infants with NEC, it is unknown whether they play a critical function in preventing NEC. This study investigated Treg development in newborn rat pups and their role in experimental NEC induction. Utilizing an established rat model of experimental NEC, the ontogeny of T cells and Tregs in newborn pups was characterized by flow cytometry. To investigate the functions of Tregs, newborn pups were given Tregs harvested from adult rats prior to NEC induction to assess clinical improvement and mechanisms of immune regulation. The results revealed that there were few Treg numbers in the terminal ileums of newborn rats and 8-fold reduction after NEC. Adoptive transfer of Tregs significantly improved weight loss, survival from 53% to 88%, and NEC incidence from 87% to 35%. The Tregs modulated the immune response as manifested in reduced CD80 expression on antigen presenting cells and decreased T cell activation within the mesenteric lymph nodes. These findings suggest that while Tregs are present in the intestines, their numbers might be insufficient to dampen the excessive inflammatory state in NEC. Adoptive transfer of Tregs attenuates the severity of NEC by limiting the immune response. Strategies to enhance Tregs have a therapeutic potential in controlling the development of NEC.</p></div
Effect of <i>Lactobacillus reuteri</i> DSM 17938 on survival, CD3<sup>+</sup> T cells and Foxp3<sup>+</sup> T cells in the ileum of rats with NEC.
<p>(a) Survival (%) of different groups of rat pups (dam-fed, NEC, and NEC+17938). (b) Changes of % CD3<sup>+</sup> T cells of lymphocytes in different groups. (c) Immunohistochemical staining of CD3<sup>+</sup> cells in rat pup ileum, 200 × magnification. (d) Number of CD3<sup>+</sup> T cells per 5 villi in IHC. (e) Percentage of CD4<sup>+</sup>Foxp3<sup>+</sup>Tregs in each group on day 4 of NEC. (f) Percentage of double positive CD4<sup>+</sup>CD8<sup>+</sup>Foxp3<sup>+</sup> T cells. The numbers represent percent of T cells (means ± SE). Each dot represents one animal in each group in the figures b, e, and f. Dam-fed: N = 15, NEC: N = 13, NEC+17938: N = 20.</p
Effect of breast milk or formula feeding with or without <i>Lactobacillus reuteri</i> DSM 17938 on the frequency of Foxp3<sup>+</sup> T cells in the ileum and mesenteric lymph node (MLN) of rats during DOL 0 and DOL 4.
<p>Newborn rats were fed immediately after birth for 4 days. (a) and (c): Changes of single CD4<sup>+</sup>Foxp3<sup>+</sup> Tregs (%) in the ileum (a) and MLN (c); (b) and (d): changes of double CD4<sup>+</sup>CD8<sup>+</sup>Foxp3<sup>+</sup> T cells in the ileum (b) and MLN (d). The percentage (%) of cells is represented as the mean ± SE of N = 9 observations (pups) in each DOL of each feeding group (total N = 45 for each feeding group).* Dam-fed +17938 vs. Dam-fed, **p<0.01, ***p<0.001. # Formula vs. Dam-fed, # p<0.05, ## p<0.01. &: Formula-fed +17938 vs. Formula-fed, & p<0.05, && p<0.01.</p
Tregs attenuated NEC induction by limiting T cell activation.
<p>(A) Representative FACS plots of CD25 on CD4<sup>+</sup>FoxP3<sup>−</sup> T cells and total percentages within the MLN at day 3 of NEC induction between NEC+Teffs (n = 10) and NEC+Tregs (n = 8) groups. (B) Representative FACS plots of CD62L expression on CD4<sup>+</sup> and CD8<sup>+</sup> T cells at day 3 of NEC induction and the percentages of CD62L<sup>+</sup> over the 3 day course within the MLN of NEC+Teffs (n = 5, 4, 10 for day 1, 2, 3 respectively) and NEC+Tregs (n = 5, 4, 8 for day 1, 2, 3 respectively). Asterisks indicate p<0.05. (C) Representative histograms of CD80 and CD86 expression on CD11b/c<sup>+</sup>RT1D<sup>−</sup> (shaded) and CD11b/c<sup>+</sup>RT1D<sup>+</sup> cells from NEC+Teffs (solid) and NEC+Tregs (dashed) groups. Bar graph is cumulative data of CD80 expression on CD11b/c<sup>+</sup>RT1D<sup>+</sup> cells from NEC+Teffs (n = 10) and NEC+Tregs (n = 8).</p
The ontogeny of T cells and Tregs in dam fed control rat pups.
<p>(A) Representative flow cytometric plots from terminal ileum on DOL 4 with initial gating on CD3<sup>+</sup> T cells to determine the percentages of CD4<sup>+</sup> and CD8<sup>+</sup> T cells (top plot) and FoxP3<sup>+</sup> Tregs within CD8<sup>−</sup>CD4<sup>+</sup> T cells (bottom plot). Frequency of (B) CD4<sup>+</sup>, (C) CD8<sup>+</sup> and (D) Tregs within the spleen (SPL), thymus (THY), mesenteric lymph nodes (MLN) and terminal ileum (INT). Absolute cell counts of CD8<sup>+</sup>, CD4<sup>+</sup>, and Tregs in the (E) MLN and (F) terminal ileums on DOL 4. Data are derived from n = 6–16 pups at each time point for B–D and n = 6 for E–F.</p
Cellularity after Treg adoptive immunotherapy.
<p>Quantitative Treg cell counts within the (A) MLN and (B) INT among the DAM and surviving pups in the NEC and NEC+Tregs groups. Quantitative numbers of CD8<sup>+</sup> and CD4<sup>+</sup> T cells within the (C) MLN and (D) terminal ileums. Data are combined three independent experiments with DAM = 26, NEC = 20 and NEC+Tregs = 23.</p