The cellular entry of Hantaan virus (HTN) occurs through interactions with beta(3) integrins as cellular receptors. However, the process of HTN infection following attachment to the cell surface is not well understood. Our data indicate that overexpression of a dominant-negative mutant dynamin inhibits HTN internalization and that compounds that block clathrin- but not caveolae-dependent endocytosis also reduce HTN infectivity. In addition, we show that HTN colocalizes with the clathrin heavy chain but not with caveolae. At the early phase of infection HTN colocalizes with EEA-1, an early endosome marker, and later, HTN colocalizes with LAMP-1, a lysosome marker. Cells treated with lysosomotropic agents are largely resistant to infection, suggesting that a low-pH-dependent step is required for HTN infection. These findings demonstrate that HTN enters cells via the clathrin-coated pit pathway and uses low-pH-dependent intracellular compartments for infectious entry.

Objective. Poly(lactic-co-glycolic acid) (PLGA), a biodegradable polymer, is a carrier for drug delivery systems. This study was undertaken to investigate the tolerogenic effect of single administration of PLGA entrapping type II collagen (CII) on the development of collagen-induced arthritis (CIA).Methods. The biophysical properties of PLGA nanoparticles entrapping CII (PLGA-CII) were investigated by in vitro release testing of CII, immunohistochemistry analysis, and electron microscopy. PLGA-CII was fed singly to animals 14 days before immunization, and the effect on joint inflammation was assessed. Circulating IgG anti-CII antibodies and T cell responses to CII in draining lymph nodes were assayed by enzyme-linked immunosorbent assay and 3 H-thymidine incorporation assay, respectively. The expression of messenger RNA (mRNA) for transforming growth factor ␤ (TGF␤) and tumor necrosis factor ␣ (TNF␣) was determined by reverse transcriptase-polymerase chain reaction.Results. The in vitro release test showed that CII was slowly discharged from PLGA-CII over a period of a month. After single administration of PLGA-CII, numerous particles ϳ300 nm in size were detectable in Peyer's patches, by electron microscopy and immunohistochemical staining for CII, 14 days after the original feeding. Mice fed a single dose of PLGA containing 40 g of CII had significantly reduced values for incidence and severity of arthritis, serum IgG anti-CII antibodies, and CII-specific T cell proliferation as compared with mice fed solvent alone, those fed 6 doses of 20 g CII alone, and those fed a single dose of PLGA alone. PLGA-CII was also able to suppress CIA after disease onset. Moreover, PLGA-CII-fed mice showed a higher level of TGF␤ mRNA expression in Peyer's patches, but a lower level of TNF␣ mRNA expression in draining lymph nodes, compared with the other groups of mice.Conclusion. Our data show that PLGA may serve as a powerful vehicle to promote the tolerance effect of oral CII and that single administration of PLGA-CII may hold promise as a new treatment strategy in rheumatoid arthritis.

Embryonic stem (ES) cells have the potential to differentiate into all three germ layers, providing new perspectives not only for embryonic development but also for the application in cell replacement therapies. Even though the formation of an embryoid body (EB) in a suspension culture has been the most popular method to differentiate ES cells into a wide range of cells, not much is known about the characteristics of EB cells. To this end, we investigated the process of EB formation in the suspension culture of ES cells at weekly intervals for up to 6 weeks. We observed that the central apoptotic area is most active in the first week of EB formation and that the cell adhesion molecules, except β-catenin, are highly expressed throughout the examination period. The sequential expression of endodermal genes in EBs during the 6-week culture correlated closely with that of normal embryo development. The outer surface of EBs stained positive for α-fetoprotein and GATA-4. When isolated from the 2-week-old EB by trypsin treatment, these endodermal lineage cells matured in vitro into hepatocytes upon stimulation with various hepatotrophic factors. In conclusion, our results demonstrate that endodermal cells can be retrieved from EBs and matured into specific cell types, opening new therapeutic usage of these in vitro differentiated cells in the cell replacement therapy of various diseases. Stem Cells 2005;23:817-827

Objective. To investigate the effector function exerted by type II collagen (CII)-stimulated T cells on rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS), and to determine their contribution to RA pathogenesis.Methods. We used enzyme-linked immunosorbent assays to measure the levels of interleukin-15 (IL-15), tumor necrosis factor ␣ (TNF␣), and IL-18 production by FLS that were cocultured with antigen-activated T cells. Likewise, we analyzed the levels of interferon-␥ (IFN␥) and IL-17 production by RA T cells coincubated with FLS. To investigate the cross-talk between CIIstimulated T cells and RA FLS, we examined the effect of using a transwell membrane to separate T cells and FLS in a culture chamber, as well as the effect of adding an antibody to block CD40 ligation.Results. The levels of IL-15, TNF␣, IFN␥, and IL-17 were all significantly increased in the serum of RA patients compared with normal control serum. Among the patients, the group with a stronger T cell proliferation response to CII showed higher levels of these

Objective. Although oral tolerance is a well-known phenomenon, the role of dendritic cells (DCs) is not well characterized. This study was conducted to better understand the differential role played by each Peyer's patch DC subset in the induction of oral tolerance to type II collagen (CII) in murine collagen-induced arthritis (CIA).Methods. CII was fed 6 times to DBA/1 mice beginning 2 weeks before immunization, and the effect on arthritis was assessed. We compared the proportion of CD11c؉,CD11b؉ DCs and CD11c؉,CD8␣؉ DCs in the Peyer's patches of CII-fed tolerized and phosphate buffered saline-fed nontolerized mice after the induction of CIA. The immunosuppressive properties of each DC subset were determined using fluorescenceactivated cell sorter analysis for intracellular interleukin-10 (IL-10) and IL-12 and mixed lymphocyte culture. The ability of each DC subset to induce CD4؉,CD25؉ T regulatory cells was also examined. Mice were injected with CII-pulsed CD11c؉,CD11b؉ DCs isolated from Peyer's patches of tolerized mice, and the effect on CIA was examined.Results. The severity of arthritis was significantly lower in tolerized mice. The proportion of CD11c؉,CD11b؉ DCs was increased in the Peyer's patches of tolerized mice and those DCs exhibited immunosuppressive characteristics, such as increased IL-10 production, inhibition of T cell proliferative responses to CII, and CD4؉,CD25؉ regulatory T cell induction. Furthermore, the CD11c؉,CD11b؉ DCs suppressed the severity of arthritis upon adoptive transfer.Conclusion. Our observations demonstrate that CD11c؉,CD11b؉ DCs, which are abundant in Peyer's patches during the induction of oral tolerance to CII, are crucial for the suppression of CIA and could be exploited for immunotherapy of autoimmune diseases.Oral tolerance refers to the immunologic hyporesponsiveness provoked by repeated exposure of the mucosal immune system to ingested protein antigens. Oral administration of antigens or peptides that are structurally similar to the autoantigen leads to local and systemic priming and, usually, to systemic tolerance, making this a promising approach for the treatment of autoimmune diseases. In animal models, it has been shown that oral tolerance effectively ameliorates experimental autoimmune encephalitis and collagen-induced arthritis (CIA) (1-8). Repeated oral administration of type II collagen (CII) induces peripheral immune tolerance, resulting in the suppression of CIA, a representative experimental model of human rheumatoid arthritis