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There have been several reports that TNF-related apoptosis-inducing ligand (TRAIL) has the ability to suppress the development of experimental autoimmune diseases, including a mouse model of experimental autoimmune encephalomyelitis, a rabbit model of rheumatoid arthritis, type 1 diabetes mellitus, in mice and experimental autoimmune thyroiditis (EAT) in mice. However, the mechanism underlying TRAIL effect is not well defined. In the present study, we specifically examined TRAIL effects on CD4(+)CD25(+) regulatory T cells. CD4(+)CD25(+) T cells prepared from mouse thyroglobulin (mTg)-immunized CBA/J mice proliferate in the presence of TRAIL and dendritic cells in vitro. These CD4(+)CD25(+) T cells included both CD4(+)CD25(+)CD45RB(Low) (regulatory) and CD4(+)CD25(+)CD45RB(High) (effector) T cells. Our results demonstrated that mTg-immunized mice treated with TRAIL showed significant increases in the number of CD4(+)CD25(+)CD45RB(Low) T cells compared with mice immunized with mTg alone. CD4(+)CD25(+)CD45RB(Low) T cells expressed much higher levels of the forkhead family transcription factor, IL-10, and TGFbeta1 than CD4(+)CD25(+)CD45RB(High) T cells, and these cells can completely suppress the proliferation of the mTg-primed splenocytes in lower concentrations than the unfractionated CD4(+)CD25(+) T cells. Furthermore, transfer of these cells into CBA/J mice prior to mTg-primed splenocyte injection could markedly reduce the frequency and severity of EAT development. CD4(+)CD25(+)CD45RB(Low) T cells were more effective at suppressing histological thyroiditis than unfractionated cells. These results indicated that TRAIL can increase the number of mTg-specific CD4(+)CD25(+)CD45RB(Low) T cells, inhibiting autoimmune responses and preventing the progression of EAT. These findings reveal a novel mechanism by which TRAIL could inhibit autoimmune disease.

Non-invasive diagnosis and detection of early-stage tumorsis regarded as one of the current challenges in the biomedicalsciences. Magnetic resonance (MR) imaging is a powerful,non-invasive imaging technique because of its high spatialresolution and tomographic capabilities. However, the signalsensitivity of MR imaging for specific biological targets islargelydependentonthespecificityandselectivityoftheligandused to target magnetic nanoparticles (NPs) to specific tissues.Development of tumor-targeted magnetic NPs is necessary toenhance the MR signal sensitivity for in-vivo tumor detection.Variousproteinssuchastransferrin

We present a general approach for the targeting and imaging of cancer cells using dendrimer-entrapped gold nanoparticles (Au DENPs). Au DENPs were found to be able to covalently link with targeting and imaging ligands for subsequent cancer-cell targeting and imaging. The Au DENPs linked with defined numbers of folic acid (FA) and fluorescein isothiocyanate (FI) molecules are water soluble, stable, and biocompatible. In vitro studies show that the FA- and FI-modified Au DENPs can specifically bind to KB cells (a human epithelial carcinoma cell line) that overexpress high-affinity folate receptors and they are internalized dominantly into lysosomes of target cells within 2 h. These findings demonstrate that Au DENPs may serve as a general platform for cancer imaging and therapeutics.

The role of TNF-related apoptosis-inducing ligand (TRAIL) in autoimmune thyroiditis is unclear. We used experimental autoimmune thyroiditis to clarify the contribution of TRAIL to the development of autoimmune thyroiditis. CBA/J mice were immunized with murine thyroglobulin, and spleen cells from these mice were subsequently injected into irradiated recipient CBA/J mice. One week later, the recipient mice were treated with recombinant TRAIL or a control protein. Compared with control animals, TRAIL-treated mice developed a milder form of the disease with a significant decrease in mononuclear cell infiltration in the thyroid and less thyroid follicular destruction. Furthermore, the number of apoptotic thyrocytes and also thyroglobulin-specific T helper-1 cell responses in TRAIL-treated mice was lower than that in the control animals. This study suggests that exogenous TRAIL suppresses the development of autoimmune thyroiditis via altering the function of cells involved in the immune response. These findings may contribute toward a novel treatment autoimmune thyroiditis.

Bacterial overgrowth in the stomach may occur under conditions of diminished or absent acid secretion. Under these conditions, secretion of the hormone gastrin is elevated. Alternatively, bacterial factors may directly stimulate gastrin. Consistent with this hypothesis, we found that mice colonized for 2 months with a mixed bacterial culture of opportunistic pathogens showed an increase in serum gastrin. To examine regulation of gene expression by bacterial proteins, stable transformants of AGS cells expressing gastrin or interleukin-8 (IL-8) promoters were cocultured with live organisms. Both whole-cell sonicates and a heat-stable fraction were also coincubated with the cells. A level of 108organisms per ml stimulated both the gastrin and IL-8 promoters. Heat-stable proteins prepared from these bacterial sonicates stimulated the promoter significantly more than the live organism or unheated sonicates. A 38-kDa heat-stable protein stimulating the gastrin and IL-8 promoters was cloned and found to be an OmpA-related protein. Immunoblotting using antibody to the OmpA-like protein identified anAcinetobactersp. as the bacterial species that expressed this protein and colonized the mouse stomach. Moreover, reintubation of mice with a pure culture of theAcinetobactersp. caused gastritis. We conclude that bacterial colonization of the stomach may increase serum gastrin levels in part through the ability of the bacteria to produce OmpA-like proteins that directly stimulate gastrin and IL-8 gene expression. These results implicate OmpA-secreting bacteria in the activation of gastrin gene expression and raise the possibility that a variety of organisms may contribute to the increase in serum gastrin and subsequent epithelial cell proliferation in the hypochlorhydric stomach.

Flow cytometry provides the opportunity to quantify cell populations within a total cell suspension. The quality of flow cytometry is strongly dependent on the isolation of intact viable cells. However, techniques to isolate mouse gastric cells for flow cytometry have not been evaluated. The objective of this study was to develop an effective method for isolating intact viable cells from mouse gastric tissue for flow cytometry. Cells were isolated from mouse stomach and spleen by either enzymatic separation or mechanical dissociation. A Percoll density gradient was used to separate viable cells from cellular debris. Cells were labeled with fluorescently tagged ligand or antibody and analyzed by flow cytometry. According to propidium iodide staining, there was a higher percentage of viable cells after mechanical dissociation (10-20%) compared to enzymatic separation (1%). After Percoll centrifugation there was a further increase in the percent of viable cells (50-80%). Gastrin (G), somatostatin (D), and parietal cells represented 0.6%, 3%, and 8% of the total epithelial cell population, respectively. T and B lymphocytes made up 4% and 2% in the gastric mucosa. Dissociated splenocytes were comprised of 20% T cells and 14% B cells. The ability to reliably resolve a cellular fraction that comprises only 0.6% of the input marks a substantial improvement over morphometric methods. Therefore, mechanical dissociation of the stomach followed by use of a Percoll gradient is the preferred method for isolating viable intact gastric epithelial cells for flow cytometry.

It has been suggested that Fas-mediated apoptosis plays an important role in the pathogenesis of autoimmune thyroid diseases. Our previous studies have demonstrated that normal primary thyroid epithelial cells are resistant to Fas-mediated apoptosis, but the resistance can be overcome by pretreatment with a combination of interferon-gamma (IFN-gamma) and IL-1beta. To understand the molecular mechanism responsible for the IFN-gamma/IL-1beta effects, we profiled changes in the transcription induced by these two cytokines in normal human thyroid cells, using cDNA microarrays. We found that IFN-gamma/IL-1beta showed a significant increase in apoptosis-related genes such as inducible nitric oxide synthase (iNOS), receptor-interacting protein 2 (RIP2), and caspases 10. These increases were confirmed by other methods, including real-time PCR and Western blot. Furthermore, the sensitization of primary thyroid epithelial cells to Fas-mediated apoptosis by IFN-gamma/IL-1beta was significantly blocked by a general caspase inhibitor, z-VAD, or by the combination of two specific individual caspase inhibitors. In addition, our results showed that IFN-gamma/IL-1beta enhance p38 MAPK phosphorylation and that SB 203580, a p38 MAPK inhibitor, can inhibit IFN-gamma/IL-1beta-induced p38 MAPK phosphorylation. SB 203580 also significantly prevented cytokine-induced iNOS expression and caspase activation and thus blocked Fas-mediated apoptosis of thyroid cells sensitized by IFN-gamma/IL-1beta. In conclusion, our data suggest that both p38 MAPK and iNOS are involved in IFN-gamma/IL-1beta-induced sensitization of the thyroid cells to Fas-mediated apoptosis via the activation of caspases 3, 7, and 10 and that this pathway may be further activated by BID. This hints that inflammatory cytokines regulate death-receptor-mediated apoptosis at multiple points in the process.

Functional interactions between factors bound at multiple sites on DNA often lead to a synergistic or more-than-additive transcriptional response. We previously defined a class of peptide sequences termed synergy control motifs (SC motifs) that function in multiple regulators by selectively inhibiting synergistic activity driven from multiple but not single response elements. By studying the prototypic SC motifs of the glucocorticoid receptor, we show that SC motifs inhibit transcription per se both in cis and in trans, and that a requirement for multiple contacts with DNA renders them selective for compound response elements. Notably, SC motifs are sites for SUMOylation, and the degree of modification correlates strongly with the extent of synergy control. Recruiting SUMO to the promoter either independently or as a fusion to the glucocorticoid receptor is sufficient to recapitulate the in trans and in cis inhibition by SC motifs without apparent changes in subcellular localization. Moreover, we find that the core ubiquitin fold domain of SUMO is sufficient for inhibition and that, independently of their potential for polySUMO chain formation, SUMO-2 and SUMO-3 are more effective inhibitors than SUMO-1.

We develop a facile approach to fabricating multifunctional dendrimer-stabilized gold nanoparticles (Au DSNPs) for cancer cell targeting and imaging. In this work, amine-terminated generation 5 (G5) poly(amidoamine) (PAMAM) dendrimers pre-functionalized with folic acid (FA) and fluorescein isothiocyanate (FI) are complexed with Au(III) ions, followed by acetylation of the amine groups on the dendrimer surfaces. This one-step process leads to the spontaneous formation of 6 nm-sized Au nanoparticles stabilized by multifunctional dendrimers bearing both targeting and imaging functionalities. The multifunctional Au DSNPs are characterized by UV-Vis spectrometry, 1H NMR, and transmission electron microscopy (TEM). The formed Au DSNPs are water-soluble, stable, and biocompatible. Combined flow cytometry, confocal microscopy, silver staining, and inductively coupled plasma-mass spectrometry (ICP-MS) analyses show that the FA- and FI-functionalized Au DSNPs can specifically target to cancer cells expressing high-affinity FA receptors in vitro. This approach to functionalizing Au DSNPs may be extended to other targeting molecules, providing a unique nanoplatform for targeting and imaging of a variety of biological systems.