Rotavirus is the most important cause of infantile gastroenteritis. Since in vivo mucosal responses to a rotavirus infection thus far have not been extensively studied, we related viral replication in the murine small intestine to alterations in mucosal structure, epithelial cell homeostasis, cellular kinetics, and differentiation. Seven-day-old suckling BALB/c mice were inoculated with 2 ؋ 10 4 focus-forming units of murine rotavirus and were compared to mock-infected controls. Diarrheal illness and viral shedding were recorded, and small intestinal tissue was evaluated for rotavirus (NSP4 and structural proteins)-and enterocyte-specific (lactase, SGLT1, and L-FABP) mRNA and protein expression. Morphology, apoptosis, proliferation, and migration were evaluated (immuno)histochemically. Diarrhea was observed from days 1 to 5 postinfection, and viral shedding was observed from days 1 to 10. Two peaks of rotavirus replication were observed at 1 and 4 days postinfection. Histological changes were characterized by the accumulation of vacuolated enterocytes. Strikingly, the number of vacuolated cells exceeded the number of cells in which viral replication was detectable. Apoptosis and proliferation were increased from days 1 to 7, resulting in villous atrophy. Epithelial cell turnover was significantly higher (<4 days) than that observed in controls (7 days). Since epithelial renewal occurred within 4 days, the second peak of viral replication was most likely caused by infection of newly synthesized cells. Expression of enterocyte-specific genes was downregulated in infected cells at mRNA and protein levels starting as early as 6 h after infection. In conclusion, we show for the first time that rotavirus infection induces apoptosis in vivo, an increase in epithelial cell turnover, and a shutoff of gene expression in enterocytes showing viral replication. The shutoff of enterocyte-specific gene expression, together with the loss of mature enterocytes through apoptosis and the replacement of these cells by less differentiated dividing cells, likely leads to a defective absorptive function of the intestinal epithelium, which contributes to rotavirus pathogenesis.Rotaviruses are one of the most significant causes of gastroenteritis, malnutrition, and diarrhea in young children and animals (17, 28). Mortality rates are low in developed countries, where illness is usually self-limiting (64). However, each year more than 600,000 young children die in developing countries throughout the world (26). Rotavirus infection in children is mainly restricted to the small intestinal villus epithelium, resulting in the occurrence of total villus atrophy (3). Although rotavirus can infect older children and adults, diarrheal disease is primarily observed in children under 2 years of age (16,17,28).Rotavirus-induced diarrhea is thought to be caused by a combination of factors (55), which include a reduction in epithelial surface area, replacement of mature enterocytes by immature (crypt-like) cells (43), an osmotic effect resulting from ...

Rotaviruses are the leading cause of severe viral gastroenteritis in young children. To gain insight in goblet cell homeostasis and intestinal mucin expression during rotavirus infection, 6-day-old mice were inoculated with murine rotavirus. To determine epithelial cell migration, mice were injected with BrdU just before inoculation. Small intestines were isolated at different days postinfection (dpi) and evaluated for rotavirus and goblet cell-specific gene expression. Small intestinal mucins of control and infected animals at 1, 2, and 4 dpi were isolated and tested for their capability to neutralize rotavirus infection in vitro. After inoculation, two peaks of viral replication were observed at 1 and 4 dpi. During infection, the number of goblet cells in infected mice was decreased in duodenum and jejunum, but was unaffected in the ileum. Goblet cells in infected animals accumulated at the tips of the villi. Muc2 mRNA levels were increased during the peak of viral replication at 1 dpi, whereas at other time points Muc2 and Tff3 mRNA levels were maintained at control levels. Muc2 protein levels in the tissue were also maintained, however Tff3 protein levels were strongly decreased. The number of goblet cells containing sulfated mucins was reduced during the two peaks of infection. Mucins isolated at 1 and 2 dpi from control and infected mice efficiently neutralized rotavirus infection in vitro. Moreover, mucins isolated from infected mice at 4 dpi were more potent in inhibiting rotavirus infection than mucins from control mice at 4 dpi. In conclusion, these data show that during rotavirus infection, goblet cells, in contrast to enterocytes, are relatively spared from apoptosis especially in the ileum. Goblet cell-specific Muc2 expression is increased and mucin structure is modified in the course of infection. This suggests that goblet cells and mucins play a role in the active defense against rotavirus infection and that age-dependent differences in mucin quantities, composition, and/or structure alter the anti-viral capabilities of small intestinal mucins.

. Alterations in Muc2 biosynthesis and secretion during dextran sulfate sodium-induced colitis.

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We sought to correlate the characteristic changes in goblet cell morphology in the chronically inflamed large intestine of 1L10-/- mice to specific changes in goblet cell gene expression. In healthy as well as IL10-/- mice, marked differences were found among the large intestinal regions in goblet cell morphology and gene expression. The mucin Muc2, which is a major determinant of goblet cell morphology, was expressed in most goblet cells, yet only in cells staining positive for both Alcian blue and high iron diamine. TFF3 was expressed in only a small subset of goblet cells. Inflamed colon of IL10-/- mice still contained high numbers of small, hypotrophic goblet cells with similar histochemical staining and Muc2 and TFF3 expression patterns, contradicting the often reported "goblet cell depletion" in colitis. Quantitatively, the Muc2 and TFF3 levels remained relatively stabile in IL10-/- mice. Muc2 in distal IL10-/- colon contained significantly less sulfate residues than in controls, which may compromise its protective properties.

Rotavirus is an important cause of morbidity and mortality worldwide and vaccines are currently under development, with clinical trails conducted in humans worldwide. The immune responses in infant BALB/c mice were examined following oral inoculation with murine rotavirus EDIM (2610 4 focus-forming units) and with three CsCl gradient-purified fractions of heterologous simian rotavirus SA11 (standardized at 2610 6 CCID 50 ) that differed in antigen composition: fraction 1 was enriched for double-layered rotavirus particles, fraction 2 for triple-layered particles and fraction 3 consisted mainly of cell components. Diarrhoea and high IgG responses, but marginal IgA responses, were observed after inoculation with all three SA11 fractions. Virus shedding was observed in all EDIM-inoculated mice, but in none of the SA11-inoculated mice. Rotavirus-specific IgG1 : 2a ratios were similar in mice inoculated with EDIM and SA11 fraction 1, but higher for SA11 fraction 3-and lower for SA11 fraction 2-inoculated mice. A higher IgG1 : 2a ratio indicates a more Th2-like immune response. This undesirable response is apparently mostly induced by inoculation with heterologous rotavirus in the presence of abundant cell-associated and soluble rotavirus proteins, compared with infection with a more purified preparation or with homologous virus. These data show that, following inoculation with a standardized amount of infectious virus, the composition of the fraction influences the outcome of the immune responses significantly. INTRODUCTIONImmune-mediated gastrointestinal disorders, such as classic IgE-mediated food allergy (Moon & Kleinman, 1995), eosinophilic gastroenteritis (Torpier et al., 1988), allergic colitis (Sherman & Cox, 1982) and eosinophilic oesophagitis (Rothenberg et al., 2001), are occurring with increased frequency in developed countries. In all of these primarily polygenic allergic disorders, Th2-type immune responses are involved. Apparently, in an increasing number of infants, the immune system is skewed towards Th2.In the so-called 'hygiene hypothesis ' (Strachan, 1989), it has been postulated that reduced exposure of young children to common infections of childhood may lead to delayed maturation of the immune system, reflected by skewing of the Th1 : Th2 balance in favour of Th2. This skewing towards a Th2-type immune response results in an increased likelihood of developing IgE-mediated allergies in atopic individuals. Matricardi et al. (2000) suggested a role of enteric pathogens in the maturation/skewing of the immune system, following the observation that allergic diseases in 20-year-old military recruits who had a history of exposure to food and orofaecal pathogens were reduced by 60 % compared with recruits without signs of exposure (Matricardi et al., 2000(Matricardi et al., , 2002.Rotavirus is a common enteric pathogen causing infection in early childhood. Even in developed countries, the great majority of children under 5 years of age experience at least one rotavirus infection (de Wit et al., 200...

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