Hideki Nakano

Biology of mouse mammary tumor virus (MMTV)

Mouse mammary tumor viruses (MMTV) replicate in the mammary gland, appear as infectious particles in mothers milk and invade the sucking pups from the intestinal tract. The immune system is essential for MMTV in the gut to reach the mammary gland. These properties make the life cycle of MMTV unique. We review the oncologically and immunologically intriguing events caused by MMTV in relation to the life cycle of the virus.

Chinook salmon embryo (CHSE-sp) cells for fish virus research.

Dear Editor: Salmonid fish ceils adapted to grow in suspension-stirred culture, a new subline of chinook salmon embryo (CHSE-214, ATCC CRL1681) ceils, designated CHSE-sp, were currently developed as reported previously (3) and have been successfully continued to proliferate only in spinner flasks (6). However, in seeking to develop the mass cultivation of cells and viruses, there are two references dealing with the suspension culture of fish cells prior to our previous study. The first reported by de Kinkelin and Le Berre (1) demonstrated a limited period of suspension culture of dissociated eyprinid cells (epithelioma papulosum cyprini, EPC) dispersed from a monolayer and proved that suspension culture of fish cells could be useful for fish virus production such as viral haemorrhagic septicemia virus (VHSV). The second by Lidgerding (4) reported the development of the first continuous suspension cultures, originally from fish cell lines CHSE-214 and fathead minnow (FHM) cells, that were propagated in earboxy methyleellulose-containing medium. While the ability of suspension adapted CHSE-214 ceils to support the replication of infectious pancreatic necrosis virus (IPNV) was retained, quite lower levels in IPNV were produced and no confirmed replication could be detected with other viruses (infectious hematopoietic necrosis virus IHNV and VHSV). The preliminary data also indicated that the low titer of IPNV produced in the suspension culture may be due to the presence of the viscid chemical in the medium (4). Objective in this study, dealing with CHSE-sp cells which have still retained the ability of cell adhesion to static substrates the same as the original monolayer ceil line CHSE-214 (3,6), is hence focused on the evaluation of the susceptibility to pathogenic fish viruses; herpesvirus salmonis type 2 (HVS-2), infectious hematopoieric necrosis virus (IHNV) and infectious pancreatic necrosis virus (IPNV). Because of the characteristic whenever the culture of CHSE-sp cells in the suspension provides opportunity to prepare viral assay (microtiter and plaque method) plates, we first evaluated the relationship of the susceptibility to a virus at various growing stages: the initiation period, linear growing period, stationary period and degradation period in the suspension culture. By employing a typical fish virus IHNV, the titers determined by the 50% tissue culture infective dose (TCIDso) method are presented in Fig. 1 with the profile of cell growth and the amount of non-adhesive cells and debris in the suspension-stirred culture. Results indicated that CHSE-sp cells from the initiation to the plateau period in the cell growth cycle had almost the same sensitivities to IHNV, titrated as approximately 107s TCIDso/ml. In the degradation period, Day 15-30th in Fig. 1, CHSE-sp cells gradually showed lower virus titers, for example, 10 6.6 TCIDs0/ml at Day 30th, while these poor sensitivity in suspension CHSE-sp ceils related to the increasing concentrations of nonadhesive cellular protein, at more than 20 #g/ml, in the suspen-

CA2+ and suspension growth of salmonid embryo cells

Dear Editor: Since the first establishment of a piscine cell hne, RTG-2 derived from rainbow trout gonad, was reported just thirty years ago (7), most established fish ceils are propagated as a monolayer ceil line under static conditions due to a general recognition as highly anchorage-dependent (5). However, two references are now available on the establishment of continuous suspension cultures of fish ceils using suspension-agitation of a spinner flask with a rotating impeller. The difference in these respective reports is in use of culture medium: the first report was by B. C. Lidgerding (1981), who used a suspension medium (EMEM-S) containing carboxymethyl cellulose, for the prolonged survival of the suspended fish cells (CHSE214, chinook salmon embryo ceils etc.) (4); the second was our current work (1991) in which Waymouths medium MB 752/1 (6) with CaC12-free modification was employed for the development of suspension CHSE-214 cells (named CHSE-sp cells) (3). In that growth medium supplemented with 5% FBS, however, approximately 160 #M of Ca 2+ was consequently supplied from the serum fraction. Whether a certain amount of Ca 2+ did influence the propagation of the suspension culture was not determined and thus, was the aim of this study. In order to evaluate the effect of Ca 2+ on the suspension growth of CHSE-sp cells, two experiments, which were at more than 160 ttM of Ca 2 + by the addition of CaC12 or at less than 160 taM adjusted with calcium specific chelating reagent EGTA to mask the divalent cation supplied from the serum fraction (160 #M), were separately conducted in suspension culture with the Waymouths medium. Results obtained in the two experiments were expressed as concentration-dependent profile of CHSE-sp cell growth (Fig. 1). Compared with the cultures at 160 #M of Ca 2+, the concentration from the serum fraction without CaCI2 addition, the highest concentration (1000 pM), which was approximately equal value to the static culture medium (original description of Waymouths MB 752/1), suppressed cell growth to less than one-half. This inhibitory effect of calcium in the suspension culture was also observed at 500 #M (about 50% inhibition of that at 160 #M), while no obvious difference between 160 and 250 #M was detected on the evaluation of the cell population. However, microscopic examination of the growing cells after seeding in the static culture dishes showed differences in cell morphology. Naturally forming celt clumps in the suspension-agitation culture were increased in number, dependent on Ca z+ concentration, and much bigger and tighter aggregates of ceils were noted at the higher concentration of Ca z+. The difference at 250 #M compared with 160 #M was thus observed in these clumping conditions. When cell clumps at 160 and 500 #M were examined by pipetting treatment in order to observe the firmness, clumps at 160 #M easily dispersed into many single cells but not those at 500 #M. Therefore, the inhibitory effects of Ca 2+ on the suspension growth of CHSE-sp cells were due to the formation of tightly clumping cells, which could not release single celts into the suspension culture medium. By employing concentrations of EGTA of 0, 60, 100, 140, and 160 #M, the residual concentration of Ca 2+ in the medium were subsequently 160, 100, 60, 20, and 0 #M, respectively. The increase in population of cultures treated with or without EGTA showed the Ca-dependent growth profiles shown in Fig 1; the highest cell density was observed in the control culture (0/.tM EGTA) which contained 160 #M of Ca 2+ (only 5% FBS). Compared with the control culture, a slight suppression of cell growth was observed at 100 and 60 ttM of Ca 2+, and the Ca 2+ concentration required to

The Role of CCR7-Ligands in Developing Experimental Autoimmune Encephalomyelitis

Multiple sclerosis is a chronic, inflammatory, and demyelinating disease of the central nervous system characterized by the pathological infiltration of autoreactive leukocytes. Experimental autoimmune encephalomyelitis serves as a disease model for human multiple sclerosis in mouse and rat (Conlon et al., 1999). Experimental autoimmune encephalomyelitis is induced through sensitization with neuroantigens such as myelin oligodendrocyte glycoprotein that activates neuroantigen-reactive T cells in the peripheral lymphoid organs. These T cells subsequently migrate into the central nervous system and encounter endogenous neuroantigens, which reactivates them and leads to nerve demyelination. Thus, induction of encephalitogenic T cells and their migration into the central nervous system are critical for development of experimental autoimmune encephalomyelitis.