Fluorescence In Vivo Hybridization (FIVH) for Detection of Helicobacter pylori Infection in a C57BL/6 Mouse Model.

INTRODUCTION: In this study, we applied fluorescence in vivo hybridization (FIVH) using locked nucleic acid (LNA) probes targeting the bacterial rRNA gene for in vivo detection of H. pylori infecting the C57BL/6 mouse model. A previously designed Cy3_HP_LNA/2OMe_PS probe, complementary to a sequence of the H. pylori 16S rRNA gene, was used. First, the potential cytotoxicity and genotoxicity of the probe was assessed by commercial assays. Further, the performance of the probe for detecting H. pylori at different pH conditions was tested in vitro, using fluorescence in situ hybridization (FISH). Finally, the efficiency of FIVH to detect H. pylori SS1 strain in C57BL/6 infected mice was evaluated ex vivo in mucus samples, in cryosections and paraffin-embedded sections by epifluorescence and confocal microscopy. RESULTS: H. pylori SS1 strain infecting C57BL/6 mice was successfully detected by the Cy3_HP_LNA/2OMe_PS probe in the mucus, attached to gastric epithelial cells and colonizing the gastric pits. The specificity of the probe for H. pylori was confirmed by microscopy. CONCLUSIONS: In the future this methodology can be used in combination with a confocal laser endomicroscope for in vivo diagnosis of H. pylori infection using fluorescent LNA probes, which would be helpful to obtain an immediate diagnosis. Our results proved for the first time that FIVH method is applicable inside the body of a higher-order animal.

Delayed growth, motor function and learning in preterm pigs during early postnatal life.

Preterm birth interrupts normal fetal growth with consequences for postnatal growth and organ development. In preterm infants, many physiological deficits adapt and disappear with advancing postnatal age, but some may persist into childhood. We hypothesized that preterm birth would induce impaired organ growth and function during the first postnatal week in pigs, while motor abilities and behavioral characteristics would show more persistent developmental delay. Cesarean-delivered preterm (n = 112, 90% gestation) or term (n = 56, 100% gestation) piglets were reared under identical conditions and euthanized for blood and organ collection on postnatal days 0, 5, or 26. Body weight gain remained lower in preterm vs. term pigs up to day 26 (25.5 ± 1.5 vs. 31.0 ± 0.5 g·kg(-1)·day(-1), P < 0.01) when relative weights were higher for brain and kidneys and reduced for liver and spleen. Neonatal preterm pigs had reduced values for blood pH, Po2, glucose, lactate, hematocrit, and cortisol, but at day 26, most values were normalized, although plasma serotonin and IGF 1 levels remained reduced. Preterm pigs showed delayed neonatal arousal and impaired physical activity, coordination, exploration, and learning, relative to term pigs (all P < 0.05). Supplementation of parenteral nutrition during the first 5 days with an enteral milk diet did not affect later outcomes. In preterm pigs, many physiological characteristics of immaturity disappeared by 4 wk, while some neurodevelopmental deficits remained. The preterm pig is a relevant animal model to study early dietary and pharmacological interventions that support postnatal maturation and neurodevelopment in preterm infants.

Breast milk contains relevant neurotrophic factors and cytokines for enteric nervous system development.

Breast-feeding plays an important role for the development of the newborn. Non-breast fed premature born infants show a significantly higher risk of developing diseases like infantile diarrhoea and necrotizing enterocolitis. In this study, the content of neurotrophic factors and cytokines, which might influence the postnatal development of the enteric nervous system (ENS), was determined in human breast milk. Glial cell-line-derived neurotrophic factor (GDNF), ciliary neurotrophic factor (CNTF) as well as a panel of cytokines were analyzed using single factor or multiplex ELISA. In order to link their presence in milk with possible effects on the development of the ENS, rat myenteric neurons were cultured in protein extracts from breast milk. Neurite outgrowth, neuron survival and nestin expression in glial cells were measured. Growth factors and cytokines were found in all breast milk samples at varying concentrations. It could be demonstrated that protein extracts of breast milk increased the amount of surviving enteric neurones as well as neurite outgrowth. Additionally it was shown, that the number of nestin and S100-expressing glial cells increased significantly after incubating in breast milk protein extracts. The data suggest that milk-born proteins support the development of the enteric nervous system.