The Impact of Linoleic Acid on Infant Health in the Absence or Presence of DHA in Infant Formulas.

Both linoleic acid (LA) and α-linolenic acid (ALA) are essential dietary fatty acids, and a balanced dietary supply of these is of the utmost importance for health. In many countries across the globe, the LA level and LA/ALA ratio in breast milk (BM) are high. For infant formula (IF), the maximum LA level set by authorities (e.g., Codex or China) is 1400 mg LA/100 kcal ≈ 28% of total fatty acid (FA) ≈ 12.6% of energy. The aims of this study are: (1) to provide an overview of polyunsaturated fatty acid (PUFA) levels in BM across the world, and (2) to determine the health impact of different LA levels and LA/ALA ratios in IF by reviewing the published literature in the context of the current regulatory framework. The lipid composition of BM from mothers living in 31 different countries was determined based on a literature review. This review also includes data from infant studies (intervention/cohort) on nutritional needs regarding LA and ALA, safety, and biological effects. The impact of various LA/ALA ratios in IF on DHA status was assessed within the context of the current worldwide regulatory framework including China and the EU. Country averages of LA and ALA in BM range from 8.5-26.9% FA and 0.3-2.65% FA, respectively. The average BM LA level across the world, including mainland China, is below the maximum 28% FA, and no toxicological or long-term safety data are available on LA levels > 28% FA. Although recommended IF LA/ALA ratios range from 5:1 to 15:1, ratios closer to 5:1 seem to promote a higher endogenous synthesis of DHA. However, even those infants fed IF with more optimal LA/ALA ratios do not reach the DHA levels observed in breastfed infants, and the levels of DHA present are not sufficient to have positive effects on vision. Current evidence suggests that there is no benefit to going beyond the maximum LA level of 28% FA in IF. To achieve the DHA levels found in BM, the addition of DHA to IF is necessary, which is in line with regulations in China and the EU. Virtually all intervention studies investigating LA levels and safety were conducted in Western countries in the absence of added DHA. Therefore, well-designed intervention trials in infants across the globe are required to obtain clarity about optimal and safe levels of LA and LA/ALA ratios in IF.

Soluble fibers and resistant starch ameliorate disease activity in interleukin-10-deficient mice with inflammatory bowel disease.

Our goal in this study was to determine the potential for dietary fibers to prevent gut inflammation in IL-10-deficient (IL-10(-/-)) mice. C57BL/6J wild-type (WT) mice (n = 90) and IL-10(-/-) mice (n = 185) were assigned to a control diet or diets supplemented with PROMITOR soluble corn fiber (SCF), STA-LITE III polydextrose (PDX), Biogum (BG), Pullulan (PI-20), PROMITOR resistant starch-75 (RS-75), SCF&BG, RS-75&BG, and inulin (4 g fiber/100 g diet). On d 47, spleen, mesenteric lymph nodes (MLN), duodenum, jejunum, ileum, and colon were macroscopically and histologically evaluated. The spleen and Peyer's patches (PP) were collected for isolating mononuclear cells and measuring the percentages of regulatory T cells (Treg) and cytokines produced by CD4(+) T cells (i.e. IFNγ and IL-10). Dietary supplementation with RS-75, SCF, RS-75&BG, and inulin ameliorated disease activity on d 47. Dietary RS-75 and inulin supplementation decreased ileal and colonic inflammatory lesions. RS-75, SCF, and inulin decreased IFNγ production by effector CD4(+) T cells from PP and RS-75 increased the IL-10-expressing cells in spleen of WT mice. Dietary SCF, PDX, BG, PI-20, and RS-75 upregulated colonic PPARγ expression in WT mice and SCF upregulated Supressor of cytokine signaling 3 in IL-10(-/-) mice. These data suggest that soluble fibers and resistant starch influence Treg cells, IFNγ, and colonic PPARγ expression to suppress gut inflammation.

Activation of PPARγ and δ by dietary punicic acid ameliorates intestinal inflammation in mice.

The goal of the present study was to elucidate the mechanisms of immunoregulation by which dietary punicic acid (PUA) prevents or ameliorates experimental inflammatory bowel disease (IBD). The expression of PPARγ and δ, their responsive genes and pro-inflammatory cytokines was assayed in the colonic mucosa. Immune cell-specific PPARγ null, PPARδ knockout and wild-type mice were treated with PUA and challenged with 2·5 % dextran sodium sulphate (DSS). The prophylactic efficacy of PUA was examined in an IL-10(-/-) model of IBD. The effect of PUA on the regulatory T-cell (Treg) compartment was also examined in mice with experimental IBD. PUA ameliorated spontaneous pan-enteritis in IL-10(-/-) mice and DSS colitis, up-regulated Foxp3 expression in Treg and suppressed TNF-α, but the loss of functional PPARγ or δ impaired these anti-inflammatory effects. At the cellular level, the macrophage-specific deletion of PPARγ caused a complete abrogation of the protective effect of PUA, whereas the deletion of PPARδ or intestinal epithelial cell-specific PPARγ decreased its anti-inflammatory efficacy. We provide in vivo molecular evidence demonstrating that PUA ameliorates experimental IBD by regulating macrophage and T-cell function through PPARγ- and δ-dependent mechanisms.

The MUC family: an obituary.

Mucins are glycoproteins that are common on the surfaces of many epithelial cells; they are deemed to mediate many interactions between these cells and their milieu. Several of these mucins form the mucus layer that is found in many hollow organs. The biophysical properties of mucins are related to their extensive O-linked glycosylation rather than directly to their polypeptide sequences. Despite the frequent absence of sequence homology, many human genes encoding mucins have been named MUC followed by a number, unjustly suggesting the existence of one large gene family. In this article, it is suggested that the mucin genes be renamed according to their sequence homologies.

Combined defects in epithelial and immunoregulatory factors exacerbate the pathogenesis of inflammation: mucin 2-interleukin 10-deficient mice.

Expression of the mucin MUC2, the structural component of the colonic mucus layer, is lowered in ulcerative colitis. Furthermore, interleukin (IL)-10 knockout (IL-10-/-) mice develop colitis and have reduced Muc2 levels. Our aim was to obtain insight into the role of Muc2 and IL-10 in epithelial protection. Muc2-IL-10 double-knockout (Muc2/IL-10(DKO)) mice were characterized and compared to Muc2 knockout (Muc2-/-), IL-10-/- and wild-type (WT) mice. Clinical symptoms, intestinal morphology and differences in epithelial-specific protein levels were analyzed. In addition, levels of the pro-inflammatory cytokines in colonic tissue and serum were determined. IL-10-/- mice were indistinguishable from WT mice throughout this experiment and showed no clinical or histological signs of colitis. Muc2/IL-10(DKO) and Muc2-/- mice showed significant growth retardation and clinical signs of colitis at 4 and 5 weeks, respectively. Muc2/IL-10(DKO) mice had a high mortality rate (50% survival/5 weeks) compared to the other types of mice (100% survival). Microscopic analysis of the colon of Muc2/IL-10(DKO) mice showed mucosal thickening, increased proliferation, superficial erosions and a diminished Muc4 expression. Furthermore, pro-inflammatory cytokines were significantly upregulated, both in tissue (mRNA) and systemically in Muc2/IL-10(DKO) mice. In conclusion, Muc2/IL-10(DKO) mice develop colitis, which is more severe in every aspect compared to Muc2-/- and IL-10-/- mice. These data indicate that (i) in case of Muc2 deficiency, the anti-inflammatory cytokine IL-10 can control epithelial damage, though to a limited extent and (ii) the mucus layer is most likely a key factor determining colitis.

The effect of Korean pine nut oil on in vitro CCK release, on appetite sensations and on gut hormones in post-menopausal overweight women.

Appetite suppressants may be one strategy in the fight against obesity. This study evaluated whether Korean pine nut free fatty acids (FFA) and triglycerides (TG) work as an appetite suppressant. Korean pine nut FFA were evaluated in STC-1 cell culture for their ability to increase cholecystokinin (CCK-8) secretion vs. several other dietary fatty acids from Italian stone pine nut fatty acids, oleic acid, linoleic acid, alpha-linolenic acid, and capric acid used as a control. At 50 muM concentration, Korean pine nut FFA produced the greatest amount of CCK-8 release (493 pg/ml) relative to the other fatty acids and control (46 pg/ml). A randomized, placebo-controlled, double-blind cross-over trial including 18 overweight post-menopausal women was performed. Subjects received capsules with 3 g Korean pine (Pinus koraiensis) nut FFA, 3 g pine nut TG or 3 g placebo (olive oil) in combination with a light breakfast. At 0, 30, 60, 90, 120, 180 and 240 minutes the gut hormones cholecystokinin (CCK-8), glucagon like peptide-1 (GLP-1), peptide YY (PYY) and ghrelin, and appetite sensations were measured. A wash-out period of one week separated each intervention day.CCK-8 was higher 30 min after pine nut FFA and 60 min after pine nut TG when compared to placebo (p < 0.01). GLP-1 was higher 60 min after pine nut FFA compared to placebo (p < 0.01). Over a period of 4 hours the total amount of plasma CCK-8 was 60% higher after pine nut FFA and 22% higher after pine nut TG than after placebo (p < 0.01). For GLP-1 this difference was 25% after pine nut FFA (P < 0.05). Ghrelin and PYY levels were not different between groups. The appetite sensation "prospective food intake" was 36% lower after pine nut FFA relative to placebo (P < 0.05). This study suggests that Korean pine nut may work as an appetite suppressant through an increasing effect on satiety hormones and a reduced prospective food intake.

Cyclooxygenase activity is important for efficient replication of mouse hepatitis virus at an early stage of infection.

Cyclooxygenases (COXs) play a significant role in many different viral infections with respect to replication and pathogenesis. Here we investigated the role of COXs in the mouse hepatitis coronavirus (MHV) infection cycle. Blocking COX activity by different inhibitors or by RNA interference affected MHV infection in different cells. The COX inhibitors reduced MHV infection at a post-binding step, but early in the replication cycle. Both viral RNA and viral protein synthesis were affected with subsequent loss of progeny virus production. Thus, COX activity appears to be required for efficient MHV replication, providing a potential target for anti-coronaviral therapy.

The effect of cytostatic drug treatment on intestine-specific transcription factors Cdx2, GATA-4 and HNF-1alpha in mice.

Chemotherapy-induced intestinal damage is a very important dose-limiting side effect for which there is no definitive prophylaxis or treatment. This is in part due to the lack of understanding of its pathophysiology and impact on intestinal differentiation. The objective of this study was to investigate the gene expression of the small intestinal transcription factors HNF-1alpha, Cdx2, GATA-4 in an experimental model of methotrexate (MTX)-induced intestinal damage, and to correlate these alterations with histological damage, epithelial proliferation and differentiation. HNF-1alpha, Cdx2 and GATA-4 are critical transcription factors in epithelial differentiation, and in combination they act as promoting factors of the sucrase-isomaltase (SI) gene, an enterocyte-specific differentiation marker which is distinctly downregulated after MTX treatment. Mice received two doses of MTX i.v. on two consecutive days and were sacrificed 1, 3 and 7 or 9 days after final injection. Segments of the jejunum were taken for morphological, immunohistochemical and quantitative analyses. Intestinal damage was most severe at day 3 and was associated with decreased expression of the transcriptional factors HNF-1alpha, Cdx2 and GATA-4, which correlated well with decreased expression of SI, and seemed inversely correlated with enhanced proliferation of epithelial crypt cells. During severe damage, the epithelium was preferentially concerned with proliferation rather than differentiation, most likely in order to restore the small intestinal barrier function rather than maintaining its absorptive function. Since HNF-1alpha, Cdx2 and GATA-4 are critical for intestine-specific gene expression and therefore crucial in epithelial differentiation, these results may explain, at least in part, why intestinal differentiation is compromised during MTX treatment.

Does small intestinal atresia affect epithelial protein expression in human newborns?

OBJECTIVES: Bowel segments distal to a congenital intestinal obstruction have been suggested to be immature. In other words, luminal components such as amniotic fluid (before birth) and/or enteral nutrition (after birth) may be required to activate intestinal epithelial protein expression, thereby influencing epithelial differentiation. We investigated cell-type-specific protein expression proximal and distal to jejunal and ileal atresias in human newborns. PATIENTS AND METHODS: We immunohistochemically studied intestinal tissue specimens of 16 newborns who had undergone surgery for jejunal or ileal atresia. Sections were taken from both the proximal and distal sides of the atresias. RESULTS: For all patients, the enterocyte-specific markers lactase, sucrase-isomaltase, sodium glucose cotransporter 1, glucose transporters 2 and 5, intestinal fatty acid-binding protein and alkaline phosphatase were expressed at a mean 3 +/- 1 days after birth, both proximal and distal to jejunal and ileal atresias. Expression of goblet cell-specific markers mucin 2 and trefoil factor 3 and that of the Paneth cell marker lysozyme was maintained at either side of the atretic segment. CONCLUSIONS: With respect to the markers used, the human small intestinal epithelium is already differentiated shortly after birth. The absence of intestinal continuity in case of a jejunal or ileal atresia does not affect epithelial protein expression. This would seem to indicate that the developing small intestinal epithelium matures independently of luminal components.

Prolonged treatment of genetically obese mice with conjugated linoleic acid improves glucose tolerance and lowers plasma insulin concentration: possible involvement of PPAR activation.

BACKGROUND: Studies in rodents and some studies in humans have shown that conjugated linoleic acid (CLA), especially its trans-10, cis-12 isomer, reduces body fat content. However, some but not all studies in mice and humans (though none in rats) have found that CLA promotes insulin resistance. The molecular mechanisms responsible for these effects are unclear, and there are conflicting reports on the effects of CLA on peroxisomal proliferator-activated receptor-gamma (PPARgamma) activation and expression. We have conducted three experiments with CLA in obese mice over three weeks, and one over eleven weeks. We have also investigated the effects of CLA isomers in PPARgamma and PPARalpha reporter gene assays. RESULTS: Inclusion of CLA or CLA enriched with its trans-10, cis-12 isomer in the diet of female genetically obese (lepob/lepob) mice for up to eleven weeks reduced body weight gain and white fat pad weight. After two weeks, in contrast to beneficial effects obtained with the PPARgamma agonist rosiglitazone, CLA or CLA enriched with its trans-10, cis-12 isomer raised fasting blood glucose and plasma insulin concentrations, and exacerbated glucose tolerance. After 10 weeks, however, CLA had beneficial effects on glucose and insulin concentrations. At this time, CLA had no effect on the plasma TNFalpha concentration, but it markedly reduced the plasma adiponectin concentration. CLA and CLA enriched with either isomer raised the plasma triglyceride concentration during the first three weeks, but not subsequently. CLA enriched with its trans-10, cis-12 isomer, but not with its cis-9, trans-11 isomer, stimulated PPARgamma-mediated reporter gene activity; both isomers stimulated PPARalpha-mediated reporter gene activity. CONCLUSIONS: CLA initially decreased but subsequently increased insulin sensitivity in lepob/lepob mice. Activation of both PPARgamma and PPARalpha may contribute to the improvement in insulin sensitivity. In the short term, however, another mechanism, activated primarily by trans-10, cis-12-CLA, which probably leads to reduced adipocyte number and consequently reduced plasma adiponectin concentration, may decrease insulin sensitivity.

Transcriptional activation of the murine Muc5ac mucin gene in epithelial cancer cells by TGF-beta/Smad4 signalling pathway is potentiated by Sp1.

The nucleotide sequence of the pMS1 clone was submitted to the GenBank Nucleotide Sequence Database under accession number AF288076. Changes in the expression of mucin genes in gastrointestinal cancers is thought to contribute to the development of the disease. In our laboratory we have shown previously that MUC5AC is aberrantly expressed in rectosigmoid villous adenomas. However, the regulatory mechanisms underlying that altered profile of expression is unknown. In order to study its regulation at the transcriptional level, we have isolated and characterized 5.5 kb of the 5'-flanking region of the mouse Muc5ac mucin gene. The promoter is flanked by a TATA box and a transcriptional start site is located 22 bp downstream of the TATA box. Analysis of the sequence showed a high density of binding sites for Smad4, an essential factor in the signalling cascade activated by TGF-beta (transforming growth factor-beta), and Sp1, an important factor in the regulation of MUC5AC. This led us to study Muc5ac regulation by TGF-beta. We show that exogenous addition of TGF-beta to the cells induces Muc5ac endogenous expression, promoter activity and Smad4 binding to the promoter. By co-transfection studies we show that Smad4 is essential for Muc5ac promoter activation and that it does not synergize with Smad2 or Smad3. By gel-retardation and co-transfection assays, we identified Sp1 and Sp3 as important regulators of Muc5ac expression and showed that Smad4 and Sp1 act in a co-operative manner to transactivate Muc5ac promoter activity. Altogether these results bring new insights into the molecular mechanisms of TGF-beta-mediated up-regulation of Muc5ac and enhance our understanding as to how Muc5ac is regulated in certain pathologies of the gastrointestinal tract.

Specific responses in rat small intestinal epithelial mRNA expression and protein levels during chemotherapeutic damage and regeneration.

The rapidly dividing small intestinal epithelium is very sensitive to the cytostatic drug methotrexate. We investigated the regulation of epithelial gene expression in rat jejunum during methotrexate-induced damage and regeneration. Ten differentiation markers were localized on tissue sections and quantified at mRNA and protein levels relative to control levels. We analyzed correlations in temporal expression patterns between markers. mRNA expression of enterocyte and goblet cell markers decreased significantly during damage for a specific period. Of these, sucrase-isomaltase (-62%) and CPS (-82%) were correlated. Correlations were also found between lactase (-76%) and SGLT1 (-77%) and between I-FABP (-52%) and L-FABP (-45%). Decreases in GLUT5 (-53%), MUC2 (-43%), and TFF3 (-54%) mRNAs occurred independently of any of the other markers. In contrast, lysozyme mRNA present in Paneth cells increased (+76%). At the protein level, qualitative and quantitative changes were in agreement with mRNA expression, except for Muc2 (+115%) and TFF3 (+81%), which increased significantly during damage, following independent patterns. During regeneration, expression of each marker returned to control levels. The enhanced expression of cytoprotective molecules (Muc2, TFF3, lysozyme) during damage represents maintenance of goblet cell and Paneth cell functions, most likely to protect the epithelium. Decreased expression of enterocyte-specific markers represents decreased enterocyte function, of which fatty acid transporters were least affected.

Interleukin 10-deficient mice exhibit defective colonic Muc2 synthesis before and after induction of colitis by commensal bacteria.

Germ-free (GF) interleukin 10-deficient (IL-10) mice develop chronic colitis after colonization by normal enteric bacteria. Muc2 is the major structural component of the protective colonic mucus. Our aim was to determine whether primary or induced aberrations in Muc2 synthesis occur in GF IL-10 mice that develop colitis after bacterial colonization. GF IL-10 and wild-type mice were colonized with commensal bacteria for various intervals up to 6 weeks. Colitis was quantified by histologic score and IL-12 secretion. Muc2 synthesis, total level of Muc2, and Muc2 sulfation were measured quantitatively. GF IL-10 mice showed 10-fold lower Muc2 synthesis and Muc2 levels compared with GF wild-type mice, but Muc2 sulfation was not different. When bacteria were introduced, IL-10 mice developed colitis, whereas wild-type mice remained healthy. Muc2 synthesis was unchanged in wild-type mice, but IL-10 mice showed a peak increase in Muc2 synthesis 1 week after bacterial introduction, returning to baseline levels after 2 weeks. Total Muc2 levels decreased 2-fold in wild-type mice but remained at stable low levels in IL-10 mice. Upon introducing bacteria, Muc2 sulfation increased 2-fold in wild-type mice, whereas in IL-10 mice Muc2 sulfation decreased 10-fold. In conclusion, a primary defect in colonic Muc2 synthesis is present in IL-10 mice, whereas bacterial colonization and colitis in these mice led to reduced Muc2 sulfation. These quantitative and structural aberrations in Muc2 in IL-10 mice likely reduce the ability of their mucosa to cope with nonpathogenic commensal bacteria and may contribute to their susceptibility to develop colitis.

Metaplasia of the duodenum shows a Helicobacter pylori-correlated differentiation into gastric-type protein expression.

The origin of gastric metaplasia of the duodenum (GMD) remains enigmatic. We studied expression of mucins and trefoil peptides in GMD to gain insight into its phenotype and origin. We examined duodenal tissue of 95 patients (0 to 83 years old, 26 with gastric Helicobacter pylori infection) for the presence of GMD. Expression was examined immunohistochemically of secretory mucins (MUC2, MUC5AC, MUC5B, and MUC6), trefoil peptides (TFF1, TFF2, and TFF3), and sucrase-isomaltase (SI). GMD, found in 37 patients, correlated positively to gastric H. pylori infection, age, and villus atrophy. MUC2 and TFF3, expressed in normal goblet cells, were absent from 100% and 87% of GMD, respectively. GMD ubiquitously expressed MUC5AC, whereas MUC5AC expression in adjacent goblet cells was closely correlated with the extent of GMD. TFF1, TFF2, and MUC6 were found in 84%, 92%, and 65% of GMD, respectively. MUC5B was absent from epithelium and GMD. SI, expressed by villus enterocytes, was absent from GMD. Brunner's glands ubiquitously expressed MUC5B, MUC6, and TFF2. GMD was characterized by the expression of gastric-type proteins MUC5AC, MUC6, TFF1, and TFF2 and the absence of intestinal markers MUC2, TFF3, and SI. In terms of the location of metaplastic cells, our results suggest that epithelial cells migrating toward villus tips switch to gastric-type secretory cells. Positive correlation with infection suggests an inductive role H. pylori in the development of GMD.

Barrett's esophagus is characterized by expression of gastric-type mucins (MUC5AC, MUC6) and TFF peptides (TFF1 and TFF2), but the risk of carcinoma development may be indicated by the intestinal-type mucin, MUC2.

Barrett's esophagus (BE) consists of metaplastic epithelium of the esophagus, generally diagnosed by mucin histochemistry. We aimed to determine which mucins were expressed in BE, and to relate their expression to BE pathology. Archival biopsies of 4 patient groups were selected, based on standard histochemistry: BE without inflammation, BE with inflammation, ulcerating BE, and BE with dysplasia. Sections were stained by immunohistochemistry for secretory mucins (MUC2, MUC5AC, MUC5B, and MUC6), the proliferation marker Ki-67, and mucin-associated trefoil factor family (TFF) peptides (TFF1, TFF2, and TFF3). MUC5AC and TFF2 were expressed at similar high levels in each clinical group. Intestinal metaplasia (IM), detected both histochemically and by the intestinal mucin MUC2, was lowest in inflamed BE. The expression of the intestinal-type TFF3 did not differ among the groups. Ulcerating BE was distinguished by very low expression of MUC6 and MUC5B, but very high expression of TFF1. Proliferation was not different among the groups. In the total group of BE patients, H. pylori infection of the stomach correlated with decreased TFF2 expression in the BE epithelium. We conclude that BE is best characterized by the specific expression of the gastric-type markers, MUC5AC, MUC6, TFF1, and TFF2. Ulcerating BE constitutes the most distinguished group with respect to mucin and TFF expression. Of the intestinal markers, MUC2 is very specific for IM in BE, whereas TFF3 is not a marker for IM. The low occurrence of IM in inflamed BE suggests that these patients may have the lowest risk of developing carcinoma.

Role of mucins in inflammatory bowel disease: important lessons from experimental models.

Inflammatory bowel disease (IBD) is characterized by a chronically inflamed mucosa of the gastrointestinal tract, caused by an underlying immune imbalance and triggered by luminal substances, including bacteria. Mucus forms a gel layer covering the gastrointestinal tract, acting as a semi-permeable barrier between the lumen and the epithelium. Mucins, the building blocks of the mucus gel, determine the thickness and properties of mucus. In IBD in humans, alterations in both membrane-bound and secretory mucins have been described involving genetic mutations in mucin genes, changes in mucin mRNA and protein levels, degree of glycosylation, sulphation, and degradation of mucins. As mucins are strategically positioned between the vulnerable mucosa and the bacterial contents of the bowel, changes in mucin structure and/or quantity probably influence their protective functions and therefore constitute possible aetiological factors in the pathogenesis of IBD. This hypothesis, however, is difficult to prove in humans. Animal models for IBD permit detailed analysis of those aspects of mucins necessary for protection against disease. These models revealed pertinent data as for how changes in mucins, in particular in MUC2, imposed by immunological or microbial factors, may contribute to the development and/or perpetuation of chronic IBD, and shed some light on possible strategies to counteract disease.

In vitro neutralisation of rotavirus infection by two broadly specific recombinant monovalent llama-derived antibody fragments.

Rotavirus is the main cause of viral gastroenteritis in young children. Therefore, the development of inexpensive antiviral products for the prevention and/or treatment of rotavirus disease remains a priority. Previously we have shown that a recombinant monovalent antibody fragment (referred to as Anti-Rotavirus Proteins or ARP1) derived from a heavy chain antibody of a llama immunised with rotavirus was able to neutralise rotavirus infection in a mouse model system. In the present work we investigated the specificity and neutralising activity of two llama antibody fragments, ARP1 and ARP3, against 13 cell culture adapted rotavirus strains of diverse genotypes. In addition, immunocapture electron microscopy (IEM) was performed to determine binding of ARP1 to clinical isolates and cell culture adapted strains. ARP1 and ARP3 were able to neutralise a broad variety of rotavirus serotypes/genotypes in vitro, and in addition, IEM showed specific binding to a variety of cell adapted strains as well as strains from clinical specimens. These results indicated that these molecules could potentially be used as immunoprophylactic and/or immunotherapeutic products for the prevention and/or treatment of infection of a broad range of clinically relevant rotavirus strains.

Alterations in epithelial and mesenchymal intestinal gene expression during doxorubicin-induced mucositis in mice.

In the current study we aimed to gain insight into epithelial-mesenchymal cross-talk and progenitor compartment modulation during doxorubicin (DOX)-induced mucositis in mice. Intestinal segments were collected on various days after DOX treatment. DOX-induced damage at day 1-2 was characterized by increased epithelial proliferation and apoptosis and a decrease in the expression of epithelial differentiation markers. Concurrently, T-cell factor-4 (TCF4) levels increased and the epithelial differentiation enhancing factor, bone morphogenic protein-4 (BMP4), decreased. During severe damage (day 3), BMP4 levels were significantly increased, which inversely correlated with epithelial proliferation. At the same time, the expression of the epithelial differentiation markers was increasing again. At day 7, BMP4 levels were down-regulated, while the levels of the epithelial differentiation markers and TCF4 were normalized again. These data suggest that in response to DOX-induced damage, BMP4 and TCF4 are modulated in such a way that homeostasis of the progenitor compartment is partly preserved.

Systemic immune response after rotavirus inoculation of neonatal mice depends on source and level of purification of the virus: implications for the use of heterologous vaccine candidates.

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 (2 x 10(4) focus-forming units) and with three CsCl gradient-purified fractions of heterologous simian rotavirus SA11 (standardized at 2 x 10(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.

Methotrexate-induced mucositis in mucin 2-deficient mice.

The mucin Muc2 or Mycin2 (Muc2), which is the main structural component of the protective mucus layer, has shown to be upregulated during chemotherapy-induced mucositis. As Muc2 has shown to have protective capacities, upregulation of Muc2 may be a counter reaction of the intestine protecting against mucositis. Therefore, increasing Muc2 protein levels could be a therapeutic target in mucositis prevention or reduction. Our aim was to determine the role of Muc2 in chemotherapy-induced mucositis. Mucositis was induced in Muc2 knockout (Muc2(-/-)) and wild type (Muc2(+/+)) mice by injecting methotrexate (MTX). Animals were weighed and sacrificed on Days 2-6 after MTX treatment and jejunal segments were analyzed. Before MTX treatment, the small intestine of Muc2(+/+) and Muc2(-/-) mice were similar with respect to epithelial morphology and proliferation. Moreover, sucrase-isomaltase and trefoil factor-3 protein expression levels were comparable between Muc2(+/+) and Muc2(-/-) mice. Up to Day 3 after MTX treatment, percentages of weight-loss did not differ. Thereafter, Muc2(+/+) mice showed a trend towards regaining weight, whereas Muc2(-/-) mice continued to lose weight. Surprisingly, MTX-induced intestinal damage of Muc2(-/-) and Muc2(+/+) mice was comparable. Prior to MTX-injection, tumor necrosis factor-alpha and interleukin-10 mRNAs were upregulated in Muc2(-/-) mice, probably due to continuous exposure of the intestine to luminal antigens. Muc2 deficiency does not lead to an increase in chemotherapy-induced mucositis. A possible explanation is the mechanism by which Muc2 deficiency may trigger the immune system to release interleukin-10, an anti-inflammatory cytokine before MTX-treatment.