Sialic acid dependence and independence of group A rotaviruses.

We have found (1), in contrast to previous reports, the human rotavirus Wa strain is sialic acid-dependent for binding to and infectivity of MA-104 cells and (2), a dual carbohydrate binding specificity is associated with both human Wa and Porcine OSU rotaviruses. One carbohydrate binding activity is associated with triple-layered virus particles (TLP) and the other with double-layered virus particles (DLP). In binding and infectivity studies, we found that gangliosides were the most potent inhibitors of both the human and procine rotavirus TLP. Furthermore, glycosylation mutant cells deficient in sialylation or neuraminidase-treated MA104 cells, did not bind rotavirus TLP from either strain. Our results show that human Wa binding and infectivity cannot be distinguished from the porcine OSU strain and appears to be sialic acid-dependent. Direct binding of human or porcine TLP to a variety of intact gangliosides was demonstrated in an thin-layer chromatographic (TLC) overlay assay. Human or porcine rotavirus DLP did not bind to any of the intact gangliosides but surprisingly bound asialogangliosides. This binding was abolished by prior treatment of the glycolipids with ceramide glycanase suggesting the intact asialoglycolipid was required for DLP binding. After treatment of either human or porcine TLP with EDTA to remove the outer shell, virus particles bound only to the immobilized asialogangliosides. These results suggest that rotavirus sugar binding specificity can be interpreted either as sialic acid-dependent or independent based on whether the virus preparation consists primarily of triple-layered or double-layered particles. Of perhaps greater interest is the possibility that sialic acid-independent carbohydrate binding activity plays a role in virus maturation or assembly.

Augmented macrophage PGE2 production following exposure to dimethylnitrosamine in vivo: relevance to suppressed T cell responses.

Previous efforts from our laboratory have investigated the mechanisms responsible for dimethylnitrosamine (DMN)-induced suppression of T cell responses. These studies suggested that such changes in T cell activity were most likely to be due to alterations in the down-regulatory signals controlling T cell activation. Accordingly, the production of PGE2, a potent inhibitor of T cell activation, was examined in macrophages obtained from animals exposed to either DMN or vehicle in vivo. The production of PGE2 was determined in macrophages representing various stages of activation (responsive, primed and fully activated) and various stages of differentiation (CSF-1-derived or GM-CSF-derived macrophages). All peritoneal macrophages obtained from DMN-exposed animals demonstrated enhanced production of PGE2 following stimulation with either endotoxin or IFN-gamma as compared to macrophages obtained from vehicle-exposed macrophages. Moreover, the enhanced levels of PGE2 were due to increased PGE2 production rather than to shifts in the kinetics of PGE2 production and utilization. CSF-1- and GM-CSF-induced bone-marrow-derived macrophages (BMDM) produced minimal levels of PGE2, regardless of the in vitro stimulation of cells obtained from either vehicle or DMN treatment groups. Spleen cells obtained from DMN-exposed animals produced significantly higher amounts of PGE2 following endotoxin stimulation compared to control splenocytes. Splenocytes from DMN-exposed animals also demonstrated a suppressed proliferative response to the mitogen Con A. However, when splenocytes from DMN-exposed animals were co-cultured with indomethacin they demonstrated Con A-stimulated proliferative responses similar to the responses of vehicle control splenocytes. These results demonstrate that DMN exposure results in increased PGE2 production by macrophages and that this increase in PGE2 production may be responsible for suppressed T cell responses observed in DMN-exposed animals.