SlzmmsryThymic epithelial cell lines (TECs) were established from newborn C57BL/6 mice. They were classified into two types (medullary and cortical TECs) by using the monoclonal antibody (Th-3) that recognizes the meshwork structure of thymic cortical epithelial cells. Antigen-presenting activity of each TEC was determined by using ovalbumin-specific, I-Ab-restricted helper T cell lines. It was demonstrated that the medullary but not the cortical TECs functioned as antigenpresenting cells. This is the first evidence for the functional difference between the cortical and the medullary TEC.T he thymic environment is the main site of T cell maturation/differentiation (1-5). On arrival in the thymus, bone marrow-derived T cell precursors maturate into thymocytes that undergo intratbymic selection. It is generally believed that cortical thymic epithelial cells (TECs) t are the main cell type that mediate positive selection, the process of selecting clones capable of self MHC-restricted antigen recognition, whereas medullary macrophages/dendritic cells or TECs are responsible for negative sdection, the process of donal elimination of autoreactive cells (6-8). There has been, however, no direct information that accounts for the functional difference of cortical and medullary TECs. In this report we demonstrated that cortical TECs are incapable of presenting soluble antigens to mature helper T cell lines while medullary TECs can function as APC, corroborating the proposed distinct roles played by these two types of TECs in selecting T cell repertoires. Materials and MethodsMice. Male and female C57BL/6 mice were purchased from Charles River Japan Inc. (Shizuoka, Japan) and were mated and reared in our specific pathogen-free mouse colony. Establishment of TECs. Thymi obtained from newborn to2-wk-old mice were digested in PBS containing 0.25% trypsin and 0.02% EDTA, and were suspended in CaZ+-free MEM. The cell suspension (104-10 s per dish) was then plated on 4,000-rad irradi-1 Abbreviation used in this paper: TEC, thymic epithelial cell.ated Swiss 3T3 cells (7 x 10 s per dish) in 60-mm dishes (3802; Falcon Labware, Oxnard, CA) at 35~ in a CO2 (5%) incubator. The culture dishes were washed with PBS containing 0.02% EDTA to remove 3T3 ceils and thymic fibroblastic cells, and then were washed with PIE containing 0.25% trypsin and 0.02% EDTA to make a suspension of TEC. Under an inverted microscope, single or clusters of the TECs were picked up and transferred to 24-well culture plates (3847; Falcon Labware) coated with 4,000-tad irradiated Swiss 3T3 cells and cultured in CaZ+-free MEM at 37~ This cloning procedure was repeated several times to obtain TECs.Antibodies. Th-3 mouse mAb against mouse thymic cortical epithelial cells was produced in our hboratory (9). M5/114 rat mAb against mouse I-A b (10) was supplied by Dr. Uchida (NIH, Tokyo, Japan). Rabbit anti-human keratin antibody was purchased from Dakopatts (Glostrup, Denmark).lmmunocytochernistry. The established TECs were cultured on eight-hole heavy Teflon-coated sl...

We investigated the ability of a baculovirus-insect cell system to produce sialylated glycoproteins. Despite the presence of enzymes for synthesizing complex-type Nglycans, the most frequent structure of insect N-glycan is the paucimannosidic type, Man 3 GlcNAc 2 (؎Fuc). The reason for the overwhelming assembly of paucimannosidic N-glycans is not yet well understood. We hypothesized that this predominance might be due to insectspecific, Golgi-associated ␤-N-acetylglucosaminidase (GlcNAcase)-mediated removal of N-acetylglucosamine residues from the precursor N-glycan, thereby preventing its galactosylation and terminal sialylation. As we expected, the suppression of intrinsic GlcNAcase activity with a specific inhibitor, 2-acetamido-1,2-dideoxynojirimycin, allowed the accumulation of sialylated glycoproteins in the supernatants of insect cell cultures after baculoviral infection. Our observation indicates that GlcNAcasedependent depletion of N-acetylglucosamine residues from intermediate N-glycans is critical for the assembly of paucimannosidic N-glycans in insect cells and, more importantly, that insect cells (under specific conditions) retain the ability to construct sialylated N-glycans like those in mammalian cells.

Following an outbreak of classical swine fever (CSF) in Japan, 2018, CSFV JPN/1/2018 was isolated from an infected pig sample. In this study, we carried out a comparative experimental infection in pigs using this strain and the highly virulent ALD strain and compared outcomes, including clinical manifestation, virus shedding patterns and antibody responses. Although pigs inoculated orally or intramuscularly with JPN/1/2018 developed hyperthermia and had decreased leucocyte numbers, they survived for the whole experimental period and showed less severe clinical signs than those infected with the ALD strain. We confirmed the presence of characteristic multifocal infarction of the margin of the spleen that arises following infection with JPN/1/2018, albeit that this finding was not observed in all infected pigs. Both viruses efficiently spread to contact pigs in a similar manner, suggesting in transmissibility between the two strains. Viral RNAs were detected in all clinical samples, especially whole blood samples, before the pigs developed hyperthermia until at least approximately 2 weeks after inoculation. Our findings will be valuable for the investigations into epidemic events occurring in Japan and for establishing diagnostic strategies and control measures against CSF.

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