The molecular mechanisms involved in the regulation of gene expression by transforming growth factor-13 (TGF-13) have been analyzed. We show that TGF-~ specifically induces the activity of the proline-rich trans-activation domain of CTF-1, a member of the CTF/NF-I family of transcription factors. A TGF-13-responsive domain (TRD) in the proline-rich transcriptional activation sequence of CTF-I was shown to mediate TGF-13 induction in NIH-3T3 cells. Mutagenesis studies indicated that this domain is not the primary target of regulatory phosphorylations, suggesting that the growth factor may regulate a CTF-l-interacting protein. A two-hybrid screening assay identified a nucleosome component, histone H3, as a specific CTF-l-interacting protein in yeast. Furthermore, the CTF-1 trans-activation domain was shown to interact with histone H3 in both transiently and stably transfected mammalian cells. This interaction requires the TRD, and it appears to be upregulated by TGF-f~ in vivo. Moreover, point mutations in the TRD that inhibit TGF-f~ induction also reduce interaction with histone H3. In vitro, the trans-activation domain of CTF-1 specifically contacts histone H3 and oligomers of histones H3 and H4, and full-length CTF-1 was shown to alter the interaction of reconstituted nucleosomal cores with DNA. Thus, the growth factor-regulated trans-activation domain of CTF-1 can interact with chromatin components through histone H3. These findings suggest that such interactions may regulate chromatin dynamics in response to growth factor signaling.
Lactobacilli derived from the endogenous flora of normal donors are being increasingly used as probiotics in functional foods and as vaccine carriers. However, a variety of studies done with distinct strains of lactobacilli has suggested heterogeneous and strain-specific effects. To dissect this heterogeneity at the immunological level, we selected two strains of lactobacilli that displayed similar properties in vitro and studied their impact on mucosal and systemic B-cell responses in monoxenic mice. Germfree mice were colonized with Lactobacillus johnsonii (NCC 533) or Lactobacillus paracasei (NCC 2461). Bacterial loads were monitored for 30 days in intestinal tissues, and mucosal and systemic B-cell responses were measured. Although both Lactobacillus strains displayed similar growth, survival, and adherence properties in vitro, they colonized the intestinal lumen and translocated into mucosal lymphoid organs at different densities. L. johnsonii colonized the intestine very efficiently at high levels, whereas the number of L. paracasei decreased rapidly and it colonized at low levels. We determined whether this difference in colonization correlated with an induction of different types of immune responses. We observed that colonization with either strain induced similar germinal center formation and immunoglobulin A-bearing lymphocytes in the mucosa, suggesting that both strains were able to activate mucosal B-cell responses. However, clear differences in patterns of immunoglobulins were observed between the two strains in the mucosa and in the periphery. Therefore, despite similar in vitro probiotic properties, distinct Lactobacillus strains may colonize the gut differently and generate divergent immune responses.The gut-associated lymphoid tissues are highly compartmentalized. The Peyer's patches are organized lymphoid tissues in the wall of the small intestine that contain B-lymphoid follicles and interfollicular populations of T cells. The lamina propria consists of large numbers of lymphoid and myeloid cells, particularly immunoglobulin A-positive (IgA ϩ ) plasmablasts, scattered under the gut epithelium. Intraepithelial lymphocytes composed mostly of CD8 ϩ T-cell subsets are interspersed within the enterocyte monolayer. It is becoming clear that the quantity and quality of the cells that populate these compartments depend on continuous stimulation provided by the endogenous intestinal microflora (9).Early studies by Schaedler et al. (36,37) contributed to the identification of members of the indigenous gut flora of mice and described the colonization of germfree mice with monocultures of these bacteria, mainly lactobacilli, enterococci, coliforms, and clostridia. Colonization of germfree mice with different mixtures of such commensal bacteria led to the rapid appearance of IgA ϩ plasma cells in the lamina propria (13, 28). Since then, germfree mice have become the model of choice to study the impact of the microflora on gut-associated lymphoid tissue development (7, 9).It was reported by several groups (...
We investigated whether certain strains of lactic acid bacteria (LAB) could antagonize specific T-helper functions in vitro and thus have the potential to prevent inflammatory intestinal immunopathologies. All strains tested induced various levels of both interleukin-12 (IL-12) and IL-10 in murine splenocytes. In particular, Lactobacillus paracasei (strain NCC2461) induced the highest levels of these cytokines. Since IL-12 and IL-10 have the potential to induce and suppress Th1 functions, respectively, we addressed the impact of this bacterium on the outcome of CD4 ؉ T-cell differentiation. For this purpose, bacteria were added to mixed lymphocyte cultures where CD4؉ T-cells from naive BALB/c mice were stimulated weekly in the presence of irradiated allogeneic splenocytes. In these cultures, L. paracasei NCC2461 strongly inhibited the proliferative activity of CD4 ؉ T cells in a dose-dependent fashion. This was accompanied by a marked decrease of both Th1 and Th2 effector cytokines, including gamma interferon, IL-4, and IL-5. In contrast, IL-10 was maintained and transforming growth factor  (TGF-) was markedly induced in a dose-dependent manner. The bacteria were not cytotoxic, because cell viability was not affected after two rounds of stimulation. Thus, unidentified bacterial components from L. paracasei NCC2461 induced the development of a population of CD4 ؉ T cells with low proliferative capacity that produced TGF- and IL-10, reminiscent of previously described subsets of regulatory cells implicated in oral tolerance and gut homeostasis.
The gut microflora play a crucial role in several physiologic functions of the host, including maturation of the gut-associated lymphoid tissues during the first months of life. Oral administration of probiotic lactic acid bacteria (LAB) modulates the immune system of humans and some laboratory animals. This effect has never been examined in dogs; therefore, our aim was to study the capacity of a probiotic LAB to stimulate immune functions in young dogs. Puppies were allotted to two groups receiving either a control diet or a diet supplemented with 5 x 10(8) colony forming units (cfu)/d of probiotic Enterococcus faecium (SF68) from weaning to 1 y of age. Fecal and blood samples were collected from the dogs at different time points for the measurement of fecal immunoglobulin (Ig)A, circulating IgG and IgA, and the proportions of lymphoid cell subsets. Fecal IgA and canine distemper virus (CDV) vaccine-specific circulating IgG and IgA were higher in the group receiving the probiotic than in controls. There were no differences in the percentages of CD4(+) and CD8(+) T cells between the groups, but the proportion of mature B cells [CD21(+)/major histocompatibility complex (MHC) class II(+)] was greater in those fed the probiotic. These data show for the first time that a dietary probiotic LAB enhance specific immune functions in young dogs, thus offering new opportunities for the utilization of probiotics in canine nutrition.
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