S100A8 and S100A9 Are Important for Postnatal Development of Gut Microbiota and Immune System in Mice and Infants.

BACKGROUND & AIMS: After birth, the immune system matures via interactions with microbes in the gut. The S100 calcium binding proteins S100A8 and S100A9, and their extracellular complex form, S100A8-A9, are found in high amounts in human breast milk. We studied levels of S100A8-A9 in fecal samples (also called fecal calprotectin) from newborns and during infancy, and their effects on development of the intestinal microbiota and mucosal immune system. METHODS: We collected stool samples (n = 517) from full-term (n = 72) and preterm infants (n = 49) at different timepoints over the first year of life (days 1, 3, 10, 30, 90, 180, and 360). We measured levels of S100A8-A9 by enzyme-linked immunosorbent assay and analyzed fecal microbiomes by 16S sRNA gene sequencing. We also obtained small and large intestine biopsies from 8 adults and 10 newborn infants without inflammatory bowel diseases (controls) and 8 infants with necrotizing enterocolitis and measured levels of S100A8 by immunofluorescence microscopy. Children were followed for 2.5 years and anthropometric data and medical information on infections were collected. We performed studies with newborn C57BL/6J wild-type and S100a9-/- mice (which also lack S100A8). Some mice were fed or given intraperitoneal injections of S100A8 or subcutaneous injections of Staphylococcus aureus. Blood and intestine, mesenterial and celiac lymph nodes were collected; cells and cytokines were measured by flow cytometry and studied in cell culture assays. Colon contents from mice were analyzed by culture-based microbiology assays. RESULTS: Loss of S100A8 and S100A9 in mice altered the phenotypes of colonic lamina propria macrophages, compared with wild-type mice. Intestinal tissues from neonatal S100-knockout mice had reduced levels of CX3CR1 protein, and Il10 and Tgfb1 mRNAs, compared with wild-type mice, and fewer T-regulatory cells. S100-knockout mice weighed 21% more than wild-type mice at age 8 weeks and a higher proportion developed fatal sepsis during the neonatal period. S100-knockout mice had alterations in their fecal microbiomes, with higher abundance of Enterobacteriaceae. Feeding mice S100 at birth prevented the expansion of Enterobacteriaceae, increased numbers of T-regulatory cells and levels of CX3CR1 protein and Il10 mRNA in intestine tissues, and reduced body weight and death from neonatal sepsis. Fecal samples from term infants, but not preterm infants, had significantly higher levels of S100A8-A9 during the first 3 months of life than fecal samples from adults; levels decreased to adult levels after weaning. Fecal samples from infants born by cesarean delivery had lower levels of S100A8-A9 than from infants born by vaginal delivery. S100 proteins were expressed by lamina propria macrophages in intestinal tissues from infants, at higher levels than in intestinal tissues from adults. High fecal levels of S100 proteins, from 30 days to 1 year of age, were associated with higher abundance of Actinobacteria and Bifidobacteriaceae, and lower abundance of Gammaproteobacteria-particularly opportunistic Enterobacteriaceae. A low level of S100 proteins in infants' fecal samples associated with development of sepsis and obesity by age 2 years. CONCLUSION: S100A8 and S100A9 regulate development of the intestinal microbiota and immune system in neonates. Nutritional supplementation with these proteins might aide in development of preterm infants and prevent microbiota-associated disorders in later years.

Where are we heading? Challenges in evidence-based severity assessment.

Evidence-based severity assessment in laboratory animals is, apart from the ethical responsibility, imperative to generate reproducible, standardized and valid data. However, the path towards a valid study design determining the degree of pain, distress and suffering experienced by the animal is lined with pitfalls and obstacles as we will elucidate in this review. Furthermore, we will ponder on the genesis of a holistic concept relying on multifactorial composite scales. These have to combine robust and reliable parameters to measure the multidimensional aspects that define the severity of animal experiments, generating a basis for the substantiation of the refinement principle.

Zinc treatment is efficient against Escherichia coli α-haemolysin-induced intestinal leakage in mice.

Zinc homoeostasis exerts protective effects in inflammatory intestinal diseases and zinc supplementation has been successfully used for treating infectious diarrhoea. This study aimed at a characterisation of zinc effects on focal leak induction by α-haemolysin (HlyA)-producing Escherichia coli (E. coli) as protective mechanism for colitis. We conducted in vivo experiments by oral challenge of gnotobiotic mice colonised with HlyA-expressing E. coli-536. Mice were either fed a defined normal or high zinc diet to analyse effects of zinc as a therapeutic regimen. HlyA-deficient E. coli-536 mutants were used as controls. Mice infected with HlyA-producing E. coli showed impaired barrier integrity when receiving normal zinc. High zinc supplementation in HlyA-producing E. coli-infected mice reduced epithelial dysfunction as indicated by ameliorated macromolecule permeability. Reduced size of focal leaks with diminished bacterial translocation was observed as inherent mechanisms of this zinc action. In human colon cell monolayers application of zinc rescued the HlyA-dependent decline in transepithelial electrical resistance via reduction of the calcium entry into HlyA-exposed cells. Calcium-dependent cell exfoliation was identified as mechanism for focal leak induction. In conclusion, zinc supplementation protects from HlyA-induced barrier dysfunction in vivo and in vitro, providing an explanation for the protective efficacy of zinc in intestinal disorders.

Investigation of Cuprizone Inactivation by Temperature.

Animal models, such as cuprizone (bis-cyclohexanone oxaldihydrazone) feeding, are helpful to study experimental demyelination and remyelination in the context of diseases like multiple sclerosis. Cuprizone is a copper chelator, which when supplemented to the normal food of C57BL/6J mice in a concentration of 0.2% leads to oligodendroglial loss, subsequent microglia and astrocyte activation, resulting in demyelination. Termination of the cuprizone diet results in remyelination, promoted by newly formed mature oligodendrocytes. The exact mode of cuprizone's action is not well understood, and information about its inactivation and cleavage are still not available. The knowledge of these processes could lead to a better understanding of cuprizone's mode of action, as well as a safer handling of this toxin. We therefore performed experiments with the aim to inactivate cuprizone by thermal heating, since it was suggested in the past that cuprizone is heat sensitive. C57BL/6J mice were fed for 4 weeks with 0.2% cuprizone, either thermally pretreated (60, 80, 105, 121 °C) or not heated. In addition, primary rat oligodendrocytes, as a known selective toxic target of cuprizone, were incubated with 350 µM cuprizone solutions, which were either thermally pretreated or not. Our results demonstrate that none of the tested thermal pretreatment conditions could abrogate or restrict the toxic and demyelinating effects of cuprizone, neither in vitro nor in vivo. In conclusion, the current study rebuts the hypothesis of cuprizone as a heat-sensitive compound, as well as the assumption that heat exposure is a reason for an insufficient demyelination of cuprizone-containing pellets.

Nanoporous silica coatings as a drug delivery system for ciprofloxacin: outcome of variable release rates in the infected middle ear of rabbits.

HYPOTHESIS: The present study was performed to examine the impact of the release rate of ciprofloxacin from prostheses coated with nanoporous silica layers on the outcome of an acute bacterial infection of the middle ear of rabbits. BACKGROUND: Middle ear prostheses are often implanted in an infectious environment because of chronic otitis media and cholesteatoma. Bacterial colonization leads to healing disorders after surgery and may lead to the extrusion of the implants. Nanoporous silica layers appear promising as a drug delivery system for antibiotics placed on implants. Before clinical applications can be envisioned, it is necessary to find an optimal release rate. METHODS: White New Zealand rabbits were provided unilaterally with either a "slow release" or a "burst release" ciprofloxacin-containing middle ear Bioverit II prosthesis. After implantation, the middle ears were infected with a solution of Pseudomonas aeruginosa. Afterwards, animals were monitored clinically and, after 3 months, sacrificed to perform necropsy and microbiologic examinations. RESULTS: In the "slow release" group, 7 of 12 animals had to be euthanized preterm because of their poor clinical condition compared with 2 of 12 animals of the "burst release" group (p < 0.05). Clinical and microbiologic examination also showed a better outcome for animals of the burst release group. CONCLUSION: A burst release of ciprofloxacin from middle ear implants is important to combat a perioperative infection with Ps. aeruginosa in the middle ear model of the rabbit.